Recombinant mammalian monocyte chemotactic protein-1 (MCP-1) receptors (MCP-1R, CCR-2)

ABSTRACT

PCT No. PCT/US95/00476 Sec. 371 Date May 25, 1995 Sec. 102(e) Date May 25, 1995 PCT Filed Jan. 11, 1995 PCT Pub. No. WO95/19436 PCT Pub. Date Jul. 20, 1995DNAs encoding receptors for the chemokine, Monocyte Chemotactic Protein-1 (MCP-1), are disclosed. Recombinant reagents and methods for expressing the DNAs are also provided. Exemplary receptor proteins are MCP-1RA and MCP-1RB, which correspond to alternatively spliced transcripts of the human MCP-1R gene. The receptor proteins of the invention are useful in assays to identify agonists and antagonists of MCP-1.

This invention was made with Government support under Grant Nos.RO1-HL42662 and RO1-HL43322 awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of PCT/US95/00476, filedJan. 11, 1995, which is a continuation-in-part of U.S. application Ser.No. 08/182,962, filed Jan. 13, 1994, now abandoned.

This invention relates to novel cytokine receptors that mediate thechemotaxis and activation of monocytes, to the DNA sequences encodingthe receptors and to processes for obtaining the receptors and producingthem by recombinant genetic engineering techniques. The novel receptorsappear to arise via alternative splicing of the DNA sequences.

BACKGROUND OF THE INVENTION

A growing family of regulatory proteins that deliver signals betweencells of the immune system has been identified. Called cytokines, theseproteins have been found to control the growth and development, andbioactivities, of cells of the hematopoietic and immune systems.Cytokines exhibit a wide range of biological activities with targetcells from bone marrow, peripheral blood, fetal liver, and otherlymphoid or hematopoietic organs. Exemplary members of the familyinclude the colony-stimulating factors (GM-CSF, M-CSF, G-CSF,interleukin-3), the interleukins (IL-1, IL-2, IL-11), the interferons(alpha, beta and gamma), the tumor necrosis factors (alpha and beta) anderythropoietin.

Within this family of proteins, an emerging group of chemotacticcytokines, also called chemokines or intercrines, has been identified.These chemokines are basic, heparin-binding proteins that haveproinflammatory and reparative activities. They are distinguished fromother cytokines having proinflammatory and reparative activities (suchas IL-1 and platelet-derived growth factor) by their characteristicconserved single open reading frames, typical signal sequences in theN-terminal region, AT rich sequences in their C-terminal untranslatedregions, and rapidly inducible mRNA expression. See, e.g., Wolpe, FASEBJ. 3:2565-73(1989) and Oppenheim, Ann. Rev. Immunol. 9:617-48(1991).Typically, the chemokines range in molecular mass from 8-10 kD; inhumans, they are the products of distinct genes clustered on chromosomes4 and 17. All chemokines have four cysteine residues, forming twodisulfide bridges.

Two subfamilies of chemokines have been recognized, based on chromosomallocation and the arrangement of the cysteine residues. The human genesfor the α, or C-X-C, subfamily members are located on human chromosome4. In this subfamily the first two cysteines are separated by one aminoacid. The members of this subfamily, the human proteins IL-8(interleukin-8), beta TG (beta thromboglobulin), PF-4 (platelet factor4), IP-10, GRO (growth stimulating factor, also known as MGSF, melanomagrow stimulating factor) and murine MIP-2 (macrophage inhibitoryprotein-2), besides having the C-X-C arrangement of their first twocystein residues, exhibit homology in their amino acid sequences in therange of 30-50%.

In the beta subfamily, the first two cysteine residues are locatedadjacent to each other, a C-C arrangement. The human genes encoding theβ subfamily proteins are located on chromosome 17 (their mousecounterparts are clustered on mouse chromosome 11 which is thecounterpart of human chromosome 17). Homology in the beta subfamilyranges from 28-45% intraspecies, from 25-55% interspecies. Exemplarymembers include the human proteins MCP-1 (monocyte chemoattractantprotein-1), LD-78 α and β, ACT-2 and RANTES and the murine proteins JEfactor (the murine homologue of MCP-1), MIP-1α and β (macrophageinhibitory protein-1) and TCA-3. Human MCP-1 and murine JE factor exertseveral effects specifically on monocytes. Both proteins are potentchemoattractants for human monocytes in vitro and can stimulate anincrease in cytosolic free calcium and the respiratory burst inmonocytes. MCP-1 has been reported to activate monocyte-mediatedtumoristatic activity, as well as to induce tumoricidal activity. See,e.g., Rollins, Mol. and Cell. Biol. 11:3125-31(1991) and Walter, Int. J.Cancer 49:431-35(1991). MCP-1 has been implicated as an important factorin mediating monocytic infiltration of tissues inflammatory processessuch as rheumatoid arthritis and alveolitis. See, e.g., Koch, J. Clin.Invest. 90:772-79(1992) and Jones, J. Immunol. 149:2147-54(1992). Thefactor may also play a fundamental role in the recruitment ofmonocyte-macrophages into developing atherosclerotic lesions, See e.g.,Nelken, J. Clin. Invest. 88:1121-27(1991), Yla-Herttuala, Proc. Nat'l.Acad. Sci. USA 88:5252-56(1991) and Cushing, Proc. Natl. Acad. Sci. USA87:5134-38(1990).

Many of these chemokines has been molecularly cloned, heterologouslyexpressed and purified to homogeneity. Several have had their receptorscloned. Two highly homologous receptors for the C-X-C chemokine IL-8have been cloned and were shown to belong to the superfamily of Gprotein-linked receptors containing seven transmembrane-spanningdomains. See Holmes, Science 253:1278-80(1991) and Murphy, Science253:1280-83(1991). More recently, a receptor for the C-C chemokinesMIP-1α and RANTES has been molecularly cloned and shown to belong to thesame seven transmembrane-spanning receptor superfamily. See Gao, J. Exp.Med. 177:1421-27(1993) and Neote, Cell 72:415-25(1993). This receptor,which is believed to be involved with leukocyte activation andchemotaxis, exhibits varying affinity and signaling efficacy dependingon the ligand. It binds with the highest affinity and the best signalingefficacy to human MIP-1α. To MCP-1, the receptor exhibits high bindingaffinity relative to RANTES and huMIP-1β but transmits signal with lowerefficacy. See Neote, Id., at 421-22. Although pharmacology studiespredicted the existence of a specific MCP-1 receptor, and the chemokinereceptors already cloned could not account for the robust responses ofmonocytes to MCP-1, to date no specific receptor for MCP-1 has beenreported. See Wang, J. Exp. Med. 177:699-705(1993) and Van Riper, J.Exp. Med. 177:851-856(1993). The difficulty may arise at least in partfrom the fact that in the chemokine family individual receptors may ormay not bind multiple ligands, making functional sorting, tracking andidentification impractical. It has also been speculated that thereceptor members of the family may not share structural features--toaccount for why the MCP-1 receptor has to date eluded researchers. SeeEdgington, Bio/Technology II:676-81(1993).

There remains a need in the art for additional receptors to thesechemokines. There also remains a need in the art for receptors specificfor each of the C-C proteins, especially a receptor specific to MCP-1.Without a specific receptor to MCP-1, there is no practical way todevelop assays of MCP-1 binding to its receptor. The availability ofsuch assays provides a powerful tool for the discovery of antagonists ofthe MCP-1/MCP-1 receptor interaction. Such antagonists would beexcellent candidates for therapeutics for the treatment ofatherosclerosis in tumor growth suppression and in other diseasescharacterized by monocytic infiltrates such as rheumatoid arthritis andalvcolitis.

SUMMARY OF THE INVENTION

In one aspect the invention provides novel human chemokine receptorproteins MCP-1RA and MCP-1RB, which are substantially free from othermammalian proteins with which they are typically found in their nativestate. MCP-1RA and MCP-1RB are identical in amino acid sequence (SEQ IDNO:2 and SEQ ID NO:4) from the 5' untranslated region through theputative seventh transmembrane domain, but they have differentcytoplasmic tails. Hence they appear to represent alternatively splicedversion of the MCP-1 gene. The proteins may be produced by recombinantgenetic engineering techniques. They may additionally be purified fromcellular sources producing the factor constituitively or upon inductionwith other factors. They may also be synthesized by chemical techniques.One skilled in the art could apply a combination of the above-identifiedmethodologies to synthesize the factor.

Active mature MCP-1RA is an approximately 374 amino acid protein havinga predicted molecular weight for the mature protein of about 42,000daltons. Its alternatively spliced version, MCP-1RB, is an approximately360 amino acid protein having a molecular weight of about 41,000daltons. The MCP-1R proteins of this invention display high specificityfor MCP-1 when expressed in Xenopus oocytes.

Another aspect of this invention is DNA sequences (SEQ ED NO:1 and SEQID NO:3) that encode the expression of the MCP-1RA and 1RB proteins.These DNA sequences may include an isolated DNA sequence that encodesthe expression of a MCP-1R protein as described above. As used here,"isolated" means substantially free from other mammalian DNA or proteinsequences with which the subject DNA or protein sequence is typicallyfound in its native, i.e., endogenous, state. The DNA sequences codingfor active MCP-1RA and 1RB are characterized as comprising the same orsubstantially the same nucleotide sequence as in FIGS. 1 and 2 (SEQ IDNOS: 1 and 3), respectively, or active fragments thereof. The DNAsequences may include 5' and 3' non-coding sequences flanking the codingsequence. The DNA sequences may also encode an amino terminal signalpeptide. FIGS. 1 and 2 illustrate the non-coding 5' and 3' flankingsequences and a signal sequence of the MCP-1RA and 1RB sequences,respectively, isolated from the human monocytic cell line MonoMac 6 andexpressed in Xenopus oocytes.

It is understood that the DNA sequences of this invention may excludesome or all of these signal and/or flanking sequences. In addition, theDNA sequences of the present invention encoding a biologically activehuman MCP-1R protein may also comprise DNA capable of hybridizing underappropriate stringency conditions, or which would be capable ofhybridizing under such conditions but for the degeneracy of the geneticcode, to an isolated DNA sequence of FIG. 1 or FIG. 2 (SEQ ID NOS:1 and3). Accordingly, the DNA sequences of this invention may containmodifications in the non-coding sequences, signal sequences or codingsequences, based on allelic variation, species variation or deliberatemodification. Additionally, analogs of MCP-1R are provided and includetruncated polypeptides, e.g., mutants in which there are variations inthe amino acid sequence that retain biological activity, as definedbelow, and preferably have a homology of at least 80%, more preferably90%, and most preferably 95%, with the corresponding region of theMCP-1R sequences of FIG. 1 or FIG. 2 (SEQ ID NOS: 2 and 4). Examplesinclude polypeptides with minor amino acid variations from the nativeamino acid sequences of MCP-1R of FIGS. 1 and 2 (SEQ ID NOS: 2 and 4);in particular, conservative amino acid replacements. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into four families: (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine,cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids. Forexample, it is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar conservative replacement of anamino acid with a structurally related amino acid will not have a majoreffect on activity or functionality.

Using the sequences of FIG. 1 and FIG. 2 (SEQ ID NOS: 1, 2, 3 and 4) aswell as the denoted characteristics of a MCP-1R receptor molecule ingeneral, it is within the skill in the art to obtain other polypeptidesor other DNA sequences encoding MCP-1R. For example, the structural genecan be manipulated by varying individual nucleotides, while retainingthe correct amino acid(s), or varying the nucleotides, so as to modifythe amino acids, without loss of activity. Nucleotides can besubstituted, inserted, or deleted by known techniques, including, forexample, in vitro mutagenesis and primer repair. The structural gene canbe truncated at its 3'-terminus and/or its 5'-terminus while retainingits activity. For example, MCP-1RA and MCP-1RB as encoded in FIG. 1 andFIG. 2 (SEQ ID NOS:1 and 2; SEQ ID NOS:3 and 4) respectively, containN-terminal regions which it may be desirable to delete. It also may bedesirable to remove the region encoding the signal sequence, and/or toreplace it with a heterologous sequence. It may also be desirable toligate a portion of the MCP-1R sequences (SEQ ID NOS: 1 and 3),particularly that which includes the amino terminal domain to aheterologous coding sequence, and thus to create a fusion peptide withthe receptor/ligand specificity of MCP-1RA or MCP-1RB.

In designing such modifications, it is expected that changes tononconserved regions of the MCP-1R sequences (SEQ ID NOS: 1, 2, 3 and 4)will have relatively smaller effects on activity, whereas changes in theconserved regions, and particularly in or near the amino terminal domainare expected to produce larger effects. The comparison among the aminoacid sequences of MCP-1RA and 1RB (SEQ ID NOS:2 and 4), theMIP-1α/RANTES receptor (SEQ ID NO:5), the orphan receptor HUMSTSR (SEQID NO:6) and the two IL-8 receptors (SEQ ID NOS: 7 and 8), asillustrated in FIG. 4, provides guidance on amino acid substitutionsthat are compatible with receptor activity. Amino acid residues that areconserved among the MCP-1R sequences (SEQ ID NOS: 2 and 4) and at leasttwo of the other sequences (SEQ ID NOS:5, 6, 7 and 8) are not expectedto be candidates for substitution. A residue which shows conservativevariations among the MCP-1R sequences and at least two of the othersequences is expected to be capable of similar conservative substitutionof the MCP-1R sequences. Similarly, a residue which variesnonconservatively among the MCP-1R sequences and at least three of theother sequences is expected to be capable of either conservative ornonconservative substitution. When designing substitutions to the MCP-1Rsequences, replacement by an amino acid which is found in the comparablealigned position of one of the other sequences is especially preferred.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, Molecular Cloning: A Laboratory Manual, Second Edition (1989);DNA Cloning, Volumes I and II (D. N. Glover, Ed. 1985); OligonucleotideSynthesis (M. J. Gait, Ed. 1984); Nucleic Acid Hybridization (B. D.Hames and S. J. Higgins, Eds. 1984); Transcription and Translation (B.D. Hames and S. J. Higgins, Eds. 1984); Animal Cell Culture (R. I.Freshney, Ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide to Molecular Cloning (1984); the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells (J. H. Miller and M. P. Calos, Eds. 1987, Cold SpringHarbor Laboratory), Methods in Enzymology, Volumes 154 and 155 (Wu andGrossman, and Wu, Eds., respectively), (Mayer and Walker, Eds.) (1987);Immunochemical Methods in Cell and Molecular Biology (Academic Press,London), Scopes, (1987); Protein Purification: Principles and Practice,Second Edition (Springer-Verlag, N.Y.); and Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, Eds 1986). Allpatents, patent applications, and publications mentioned herein, bothsupra and infra, are hereby incorporated by reference.

Additionally provided by this invention is a recombinant DNA vectorcomprising vector DNA and a DNA sequence (SEQ ID NOS: 1 and 3) encodinga mammalian MCP-1R polypeptide. The vector provides the MCP-1R DNA inoperative association with a regulatory sequence capable of directingthe replication and expression of an MCP-1R protein in a selected hostcell. Host cells transformed with such vectors for use in expressingrecombinant MCP-1R proteins are also provided by this invention. Alsoprovided is a novel process for producing recombinant MCP-1R proteins oractive fragments thereof. In this process, a host cell line transformedwith a vector as described above containing a DNA sequence (SEQ ID NOS:1 and 3) encoding expression of an MCP-1R protein in operativeassociation with a suitable regulatory sequence capable of directingreplication and controlling expression of an MCP-1R protein is culturedunder appropriate conditions permitting expression of the recombinantDNA. The expressed protein is then harvested from the host cell orculture medium using suitable conventional means. This novel process mayemploy various known cells as host cell lines for expression of theprotein. Currently preferred cell lines are mammalian cell lines andbacterial cell lines.

This invention also provides compositions for use in therapy, diagnosis,assay of MCP-1R, or in raising antibodies to MCP-1R, comprisingeffective amounts of MCP-1R proteins prepared according to the foregoingprocesses. Another aspect of this invention provides an assay to assessMCP-1 binding, useful in screening for specific antagonists of the MCP-1receptor. Such assay comprises the steps of expression and isolation ofthe recombinant MCP-1 receptor(s) and/or their extracellular domains andthe development of a solid-phase assay for MCP-1 binding. Theavailability of such assays, not heretofore available, permits thedevelopment of therapeutic antagonists, useful in the treatment ofatherosclerosis and other diseases characterized by monocyticinfiltrates.

A further aspect of the invention therefore are pharmaceuticalcompositions containing a therapeutically effective amount of an MCP-1antagonist identified using the assays of this invention. Such MCP-1antagonist compositions may be employed in therapies foratherosclerosis, cancer and other diseases characterized by monocyticinfiltrates. An additional aspect therefore, the invention includes amethod for treating these and/or other diseases and pathological statesby administering to a patient a therapeutically effective amount ofMCP-1 antagonist, or an active fragment thereof, in a suitablepharmaceutical carrier.

Other aspects and advantages of this invention are described in thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate the human cDNA and amino acid sequences (SEQ IDNO:1 and SEQ ID NO:2, respectively) of the isolated MCP-1 receptorclone, MCP-1RA.

FIGS. 2A-2C illustrate the human cDNA and amino acid sequences (SEQ IDNO:3 and SEQ ID NO:4, respectively) of the isolated MCP-1 receptorclone, MCP-1RB.

FIGS. 3A and 3B illustrate the results of Northern blot analysis ofhematopoietic cell lines that were probed for MCP-1RA and MCP-1RB mRNA,respectively.

FIGS. 4A and 4B illustrate the predicted amino acid sequence of theMCP-1 receptor A (MCP-1RA) (SEQ ID NO:2), aligned with the MIP-1α/RANTESreceptor sequence (SEQ ID NO:5), the orphan receptor sequence HUMSTSR(SEQ ID NO:6) and the two IL-8 receptor seqeunces (SEQ ID NOS:7 and 8).Identical residues are boxed. The seven putative transmembrane domainsare indicated by the horizontal bars. Gaps inserted to optimize thealignments are indicated by dashes. Amino acid numbers for each sequenceare located to the right of the sequences.

FIG. 5 graphically depicts the functional expression of MCP-1R proteinin Xenopus oocytes as assayed by measuring calcium mobilization in thepresence of MCP-1.

FIG. 6 graphically depicts the results of the calcium efflux assay usedto confirm gene expression and responsiveness to MCP-1 as described inExample 4.

FIGS. 7A and 7B graphically depict the binding of ¹²⁵ I-MCP-1 to therecombinant MCP-1RB receptor, as described in detail in Example 5.

FIGS. 8A-8C graphically depict the results of the MCP-1RBreceptor-mediated calcium mobilization experiments also described indetail in Example 5. FIG. 8A depicts intracellular calcium flux as afunction of MCP-1 concentration (nM). Calcium transients peaked at 4-8sec. after addition of MCP-1 and returned to baseline within 90 sec. ofactivation. FIG. 8B depicts the MCP-1 stimulated calcium mobilization(EC₅₀ =3.4 nM) and the lack of stimulated calcium mobilization by othercytokines. FIG. 8C illustrates that MCP-1 desensitized the cells to asecond addition of MCP-1.

DETAILED DESCRIPTION

I. Introduction

This invention provides biologically active human chemokine receptors,MCP-1RA and 1RB, substantially free from association with othermammalian proteins and proteinaceous material with which they arenormally associated in its native state. MCP-1R proteins can be producedby recombinant techniques to enable production in large quantitiesuseful for assaying potential antagonists to identify candidates fortherapeutics for the treatment of atherosclerosis and other monocyticassociated diseases such as cancer and rheumatoid arthritis.Alternatively, MCP-1R proteins may be obtained as a homogeneous proteinpurified from a mammalian cell line secreting or expressing it, or theymay be chemically synthesized.

Human MCP-1RA was isolated from a derivative of a human monocyticleukemia cell line, MonoMac 6 (MM6). Because monocytes are difficult toisolate in large quantities and express less than 2000 high-affinitybinding sites per cell, a cell line that responded well to MCP-1 wasneeded. Because of their consistency in response, the MM6 cell line waschosen. It can be obtained from the DSM German Collection ofMicroorganisms and Cell Cultures (Mascheroder Weg1b, 3300 Braunschweig,Germany); see also, Ziegler-Heitbrock, Int. J. Cancer 41:456(1988).Cells were grown in appropriate medium and then tested for changes inintracellular calcium in response to MCP-1 and other chemokines. A cDNAlibrary was prepared from MonoMac 6 mRNA according to methods previouslydescribed. See Vu, Cell 64:1057-68(1991). A polymerase chain reaction(PCR)-based strategy using degenerate oligonucleotide primerscorresponding to conserved sequences in the second and thirdtransmembrane domains of the other chemokine receptors and in theHUMSTSR orphan receptor was employed (See SEQ ID NOS: 5, 6, 7 and 8).Amplification of cDNA derived from MM6 cells using the primers yielded anumber of PCR products corresponding in size to those expected for aseven-transmembrane receptor. Analysis of the subcloned PCR productsrevealed cDNAs encoding the predicted arrangements of the receptors uponwhich the primers were designs, along with one cDNA that appeared toencode a novel receptor.

To obtain a full-length version of this clone, an MM6 cDNA library wasconstructed and probed with the PCR product. An isolated clone of 2.1 kbwas obtained and called MCP-1RA. FIGS. 1A-1D illustrate the cDNAsequence (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ IDNO:2) of the clone. The nucleotide sequence (SEQ ID NO:1) comprises 2232base pairs, including a 5' noncoding sequence of 39 base pairs and a 3'noncoding sequence of 1071 base pairs. The MCP-1RA sequence ischaracterized by a single long open reading frame encoding a 374 aminoacid following the initiation methionine at position 23.

The nucleotide sequence of MCP-1RA cDNA (SEQ ID NO: 1) was compared withthe nucleotide sequences recorded in Genbank. Homology was found withthe coding sequences of the receptors for MIP-1 alpha/RANTES, theHUMSTSR orphan receptor and IL-8 (SEQ ID NOS: 5, 6, 7 and 8,respectively). No significant homology was found between the codingsequence of MCP-1RA and any other published polypeptide sequence.

The predicted amino acid sequence of MCP-1RA (SEQ ID NO:2) reveals sevenputative transmembrane domains and an extracellular amino terminus of 40residues. Further analysis of the MCP-1RA amino acid sequence revealsseveral interesting features. Despite its homology with the relatedMIP-1 alpha/RANTES receptor and the IL-8 receptors, MCP-1RA exhibitssignificant divergence in its amino and carboxyl termini. See FIGS.4A-4B (SEQ ID NOS: 2, 5, 6, 7 and 8). Additionally, a striking identitybetween MCP-1RA and the MIP-1 alpha/RANTES receptor occurs in a 31 aminoacid sequence beginning with the septate IFFIILL at the end of the thirdtransmembrane domain.

Preliminary biological characterization indicates that MCP-1RA confersrobust and remarkable specific responses to nanomolar concentrations ofMCP-1. Surprisingly, no response was elicited by the MIP-1α, MIP-1β,RANTES or Il-8, even at concentrations of 500 nanomoles.

Analysis of additional clones in the MM6 cDNA library revealed a secondsequence, identical to the MCP-1RA sequence from the 5' untranslatedregion through the putative seventh transmembrane domain but containinga different cytoplasmic tail. This second sequence (SEQ ID NOS:3 and 4),termed MCP-1RB, appears to be an alternatively spliced version ofMCP-1RA. It is further characterized below.

The MCP-1R polypeptides provided herein also include polypeptidesencoded by sequences similar to that of MCP-1RA and 1RB (SEQ ID NOS: 1,2, 3 and 4) in FIGS. 1A-1D and 2A-2C, but into which modifications arenaturally provided or deliberately engineered. This invention alsoencompasses such novel DNA sequences, which code on expression forMCP-1R polypeptides having specificity for the MCP-1 receptor. These DNAsequences include sequences substantially the same as the DNA sequences(SEQ ID NOS: 1 and 3) of FIGS. 1A-1D and 2A-2C and biologically activefragments thereof, and such sequences that hybridize under stringenthybridization conditions to the DNA sequences (SEQ ID NOS: 1 and 3) ofFIGS. 1A-1D and 2A-2C. See Maniatis, Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory (1982), pages 387-389. Oneexample of such stringent conditions is hybridization at 4 ×SSC, at 65degrees C., followed by a washing in 0.1×SSC at 65 degrees C. for onehour. Another exemplary stringent hybridization scheme uses 50%formamide, 4×SSC at 42 degrees C.

DNA sequences that code for MCP-1R polypeptides but differ in codonsequence due to the degeneracies inherent in the genetic code are alsoencompassed by this invention. Allelic variations, i.e., naturallyoccurring interspecies base changes that may or may not result in aminoacid changes, in the MCP-1R DNA sequences (SEQ ID NOS: 1 and 3) of FIGS.1A-1D and 2A-2C encoding MCP-1R polypeptides having MCP-1R activity (forexample, specificity for the MCP-1 receptor) are also included in thisinvention.

II. Modes for Carrying Out the Invention

Methods for producing a desired mature polypeptide can include thefollowing techniques. First, a vector coding for a MCP-1R polypeptidecan be inserted into a host cell, and the host cell can be culturedunder suitable culture conditions permitting production of thepolypeptide.

The MCP-1R genes or fragments thereof can be expressed in a mammalian,insect, or microorganism host. The polynucleotides encoding MCP-1R genesare inserted into a suitable expression vector compatible with the typeof host cell employed and is operably linked to the control elementswithin that vector. Vector construction employs techniques which areknown in the art. Site-specific DNA cleavage involved in suchconstruction is performed by treating with suitable restriction enzymesunder conditions which generally are specified by the manufacturer ofthese commercially available enzymes.

A suitable expression vector is one that is compatible with the desiredfunction (e.g., transient expression, long term expression, integration,replication, amplification) and in which the control elements arecompatible with the host cell.

A. Expression in mammalian cells

Vectors suitable for replication in mammalian cells are known in theart, and can include viral replicons, or sequences that ensureintegration of the sequence encoding MCP-1R into the host genome.Exemplary vectors include those derived from simian virus SV40,retroviruses, bovine papilloma virus, vaccinia virus, and adenovirus.

As is known in the art, the heterologous DNA, in this case MCP-1R DNA,is inserted into the viral genome using, for example, homologousrecombination techniques. The insertion is generally made into a genewhich is non-essential in nature, for example, the thymidine kinase gene(tk), which also provides a selectable marker. Plasmid shuttle vectorsthat greatly facilitate the construction of recombinant viruses havebeen described (see, for example, Mackett, et al. (1984); Chakrabarti,et al. (1985); Moss (1987)). Expression of the heterologous polypeptidethen occurs in cells or individuals which are immunized with the liverecombinant virus.

Such suitable mammalian expression vectors usually contain a promoter tomediate transcription of foreign DNA sequences and, optionally, anenhancer. Suitable promoters for mammalian cells are known in the artand include viral prompters such as that from simian virus 40 (SV40),cytomegalovirus (CMV), Rous sarcoma virus (RSV), adenovirus (ADV), andbovine papilloma virus (BPV).

The optional presence of an enhancer, combined with the promoterdescribed above, will typically increase expression levels. An enhanceris any regulatory DNA sequence that can stimulate transcription up to1000-fold when linked to endogenous or heterologous promoters, withsynthesis beginning at the normal mRNA start site. Enhancers are alsoactive when placed upstream or downstream from the transcriptioninitiation site, in either normal or flipped orientation, or at adistance of more than 1000 nucleotides from the promoter. See Maniatis,Science 236:1237(1987), Alberts, Molecular Biology of the Cell, 2nd Ed.(1989). Enhancer elements derived from viruses may be particularlyuseful, because they typically have a broader host range. Examplesuseful in mammalian cells include the SV40 early gene enhancer (seeDijkema, EMBO J. 4:761(1985)) and the enhancer/promoters derived fromthe long terminal repeat (LTR) of the RSV (see Gorman, Proc. Natl. Acad.Sci. 79:6777(1982b)) and from human cytomegalovirus (see Boshart, Cell41:521(1985)). Additionally, some enhancers are regulatable and becomeactive only in the presence of an inducer, such as a hormone or metalion (see Sassone-Corsi and Borelli, Trends Genet. 2:215(1986));Maniatis, Science 236:1237(1987)).

In addition, the expression vector can and will typically also include atermination sequence and poly(A) addition sequences which are operablylinked to the MCP-1R coding sequence.

Sequences that cause amplification of the gene may also be desirablyincluded in the expression vector or in another vector that isco-translated with the expression vector containing an MCP-1R DNAsequence, as are sequences which encode selectable markers. Selectablemarkers for mammalian cells are known in the art, and include forexample, thymidine kinase, dihydrofolate reductase (together withmethotraxate as a DHFR amplifier), aminoglycoside phosphotransferase,hygromycin B phosphotransferase, asparagine synthetase, adenosinedeaminase, metallothionien, and antibiotic resistant genes such asneomycin.

The vector that encodes an MCP-1R polypeptide can be used fortransformation of a suitable mammalian host cell. Transformation can beby any known method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus andtransducing a host cell with the virus. The transformation procedureused depends upon the host to be transformed. Methods for introductionof heterologous polynucleotides into mammalian cells are known in theart and include dextran-mediated transection, calcium phosphateprecipitation, polybrene mediated transection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,Hep G2), and a number of other cell lines.

B. Expression in Insect Cells

In the case of expression in insect cells, generally the components ofthe expression system include a transfer vector, usually a bacterialplasmid, which contains both a fragment of the baculovirus genome, and aconvenient restriction site for insertion of the heterologous gene orgenes to be expressed; a wild type baculovirus with a sequencehomologous to the baculovirus-specific fragment in the transfer vector(this allows for the homologous recombination of the heterologous genein to the baculovirus genome); and appropriate insect host cells andgrowth media.

Exemplary transfer vectors for introducing foreign genes into insectcells include pAc373 and pVL985. See Luckow and Summers, Virology17:31(1989).

The plasmid usually also contains the polyhedron polyadenylation signaland a procaryotic ampicillin-resistance (amp) gene and origin ofreplication for selection and propagation in E. coli. See Miller, Ann.Rev. Microbiol. 42:177(1988).

Baculovirus transfer vectors usually contain a baculovirus promoter,i.e., a DNA sequence capable of binding a baculovirus RNA polymerase andinitiating the downstream (5' to 3') transcription of a coding sequence(e.g., structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5' end of thecoding sequence. This transcription initiation region typically includesan RNA polymerase binding site and a transcription initiation site. Abaculovirus transfer vector can also have an enhancer, which, ifpresent, is usually distal to the structural gene. Expression can beeither regulated or constitutive.

C. Expression in Microorganisms--Yeast and Bacteria

Fungal expression systems can utilize both yeast and filamentous fungihosts. Examples of filamentous fungi expression systems are Aspergillus,as described in EP Patent Pub. No. 357 127 (published Mar. 7, 1990), andAcremonium Chrysogenum, described in EP Patent Pub. No. 376 266(published Jul. 4, 1990).

A yeast expression system can typically include one or more of thefollowing: a promoter sequence, fusion partner sequence, leadersequence, transcription termination sequence.

A yeast promoter, capable of binding yeast RNA polymerase and initiatingthe downstream (3') transcription of a coding sequence (e.g. structuralgene) into mRNA, will have a transcription initiation region usuallyplaced proximal to the 5' end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site (a"TATA Box") and a transcription initiation site. The yeast promoter canalso have an upstream activator sequence, usually distal to thestructural gene. The activator sequence permits inducible expression ofthe desired heterologous DNA sequence. Constitutive expression occurs inthe absence of an activator sequence. Regulated expression can be eitherpositive or negative, thereby either enhancing or reducingtranscription.

Particularly useful yeast promoter sequences include alcoholdehydrogenase (ADH) (EP Patent Pub. No. 284 044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK)(EP Patent Pub. No. 329 203). The yeastPHO5 gene, encoding acid phosphatase, also provides useful promotersequences. See Myanohara, Proc. Natl. Acad. Sci. USA80:1(1983).

An MCP-1R gene or an active fragment thereof can be expressedintracellularly in yeast. A promoter sequence can be directly linkedwith an MCP-1R gene or fragment, in which case the first amino acid atthe N-terminus of the recombinant protein will always be a methionine,which is encoded by the ATG start codon. If desired, methionine at theN-terminus can be cleaved from the protein by in vitro incubation withcyanogen bromide.

Intracellularly expressed fusion proteins provide an alternative todirect expression of an MCP-1R sequence. Typically, a DNA sequenceencoding the N-terminal portion of a stable protein, a fusion partner,is fused to the 5' end of heterologous DNA encoding the desiredpolypeptide. Upon expression, this construct will provide a fusion ofthe two amino acid sequences. For example, the yeast or human superoxidedismutase (SOD) gene, can be linked at the 5' terminus of an MCP-1Rsequence and expressed in yeast. The DNA sequence at the junction of thetwo amino acid sequences may or may not encode a cleavable site. See,e.g., EP Patent Pub. No. 196 056. Alternatively, MCP-1R polypeptides canalso be secreted from the cell into the growth media by creating afusion protein comprised of a leader sequence fragment that provides forsecretion in yeast or bacteria of the MCP-1R polypeptides. Preferably,there are processing sites encoded between the leader fragment and theMCP-1R sequence (SEQ ID NOS: 1 and 3) that can be cleaved either in vivoor in vitro. The leader sequence fragment typically encodes a signalpeptide comprised of hydrophobic amino acids which direct the secretionof the protein from the cell. DNA encoding suitable signal sequences canbe derived from genes for secreted yeast proteins, such as the yeastinvertase gene (EP Patent Pub. No. 12 873) and the A-factor gene (U.S.Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such asan interferon leader, can be used to provide for secretion in yeast (EPPatent Pub. No. 60057). Transcription termination sequences recognizedby yeast are regulatory regions located 3' to the translation stopcodon. Together with the promoter they flank the desired heterologouscoding sequence. These flanking sequences direct the transcription of anmRNA which can be translated into the MCP-1R polypeptide encoded by theMCP-1R DNA.

Typically, the above described components, comprising a promoter, leader(if desired), coding sequence of interest, and transcription terminationsequence, are put together in plasmids capable of stable maintenance ina host, such as yeast or bacteria. The plasmid can have two replicationsystems, so it can be maintained as a shuttle vector, for example, inyeast for expression and in a procaryotic host for cloning andamplification. Examples of such yeast-bacteria shuttle vectors includeYEp24 (see Botstein, Gene 8:17-24 (1979)), pCl/1 (see Brake, Proc. Natl.Acad. Sci. USA 81:4642-4646(1984)), and YRp17 (see Stinchcomb, J. Mol.Biol. 158:157(1982)). In addition, the plasmid can be either a high orlow copy number plasmid. A high copy number plasmid will generally havea copy number ranging from about 5 to about 200, and typically about 10to about 150. A host containing a high copy number plasmid willpreferably have at least about 10, and more preferably at least about20. Either a high or low copy number vector may be selected, dependingupon the effect on the host of the vector and the MCP-1R polypeptides.See, e.g., Brake, et al., supra.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors typicallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. See Orr-Weaver, Methods InEnzymol. 101:228-245(1983) and Rine, Proc. Natl. Acad. Sci. USA80:6750(1983).

Typically, extrachromosomal and integrating expression vectors cancontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers can include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker can also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions.See Butt, Microbiol. Rev. 51:351(1987).

Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors aretypically comprised of a selectable marker that is either maintained ina replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal orintegrating, have been developed for transformation into many yeasts.Exemplary yeasts cell lines are Candida albicans (Kurtz, Mol. Cell.Biol. 6:142(1986), Candida maltosa (Kunze, J. Basic Microbiol.25:141(1985), Hansenula polymorpha (Gleeson, J. Gen. Microbiol.132:3459(1986) and Roggenkamp, Mol. Gen. Genet. 202:302(1986),Kluyveromyces fragilis (Das, J. Bacteriol. 158:1165(1984), Kluyveromyceslactis (De Louvencourt, J. Bacteriol. 154:737(1983) and Van den Berg,Bio/Technology 8:135(1990), Pichia guillerimondii (Kunze, J. BasicMicrobiol. 25:141(1985), Pichia pastoris (Cregg, Mol. Cell. Biol. 5:3376(1985), Saccharomyces cerevisiae (Hinnen, PROC. NATL. ACAD. SCI. USA75:1929(1978) and Ito, J. Bacteriol. 153:163(1983), Schizosaccharomycespombe (Beach and Nurse, Nature 300:706(1981), and Yarrowia lipolytica(Davidow, Curr. Genet. 10:380471(1985) and Gaillardin, Curr. Genet.10:49(1985)

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and typically include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seethe publications listed in the foregoing paragraph for appropriatetransformation techniques.

Additionally, the MCP-1R gene or fragment thereof can be expressed in abacterial system. In such system, a bacterial promoter is any DNAsequence capable of binding bacterial RNA polymerase and initiating thedownstream (3') transcription of a coding sequence (e.g. a desiredheterologous gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5' end of thecoding sequence. This transcription initiation region typically includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter can also have a second domain called an operator,that can overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein can bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression can occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation can be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5') to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli). See Raibaud, Ann. Rev. Genet. 18:173(1984). Regulatedexpression can therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) (see Chang,Nature 198:1056(1977), and maltose. Additional examples include promotersequences derived from biosynthetic enzymes such as tryptophan (trp)(see Goeddel, NUC. ACIDS RES. 8:4057(1981), Yelverton, Nuc. Acids Res.9:731(1981), U.S. Pat. No. 4,738,921 and EP Patent Pub. Nos. 36 776 and121 775). The β-lactamase (bla) promoter system (see Weissmann,Interferon 3 (ed. I. Gresser), the bacteriophage lambda PL promotersystem (see Shimatake, Nature 292:128(128) and the T5 promoter system(U.S. Pat. No. 4,689,406) also provides useful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter can be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter such as the tac promoter (see U.S.Pat. No. 4,551,433, Amann, Gene 25:167(1983) and de Boer, Proc. Natl.Acad. Sci. 80:21(1983)). A bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is exemplary. (see Studier, J. Mol. Biol. 189:113(1986) andTabor, Proc. Natl. Acad. Sci. 82:1074(1985)).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of the MCP-1R gene orfragment thereof in prokaryotes. In E. coli, the ribosome binding siteis called the Shine-Dalgarno (SD) sequence and includes an initiationcodon (ATG) and a sequence 3-9 nucleotides in length located 3-11nucleotides upstream of the initiation codon (see Shine, Nature254:34(1975). The SD sequence is thought to promote binding of mRNA tothe ribosome by the pairing of bases between the SD sequence and the 3'and of E. coli 16S rRNA (see Steitz, Biological Regulation andDevelopment: Gene Expression (ed. R. F. Goldberger)(1979)).

MCP-1R protein can be expressed intracellularly. A promoter sequence canbe directly linked with an MCP-1R gene or a fragment thereof, in whichcase the first amino acid at the N-terminus will always be a methionine,which is encoded by the ATG start codon. If desired, methionine at theN-terminus can be cleaved from the protein by in vitro incubation withcyanogen bromide or by either in vivo on in vitro incubation with abacterial methionine N-terminal peptidase. See EP Patent Pub. No. 219237.

Fusion proteins provide an alternative to direct expression. Typically,a DNA sequence encoding the N-terminal portion of an endogenousbacterial protein, or other stable protein, is fused to the 5' end ofheterologous MCP-1R coding sequences. Upon expression, this constructwill provide a fusion of the two amino acid sequences. For example, thebacteriophage lambda cell gene can be linked at the 5' terminus of anMCP-1R gene or fragment thereof and expressed in bacteria. The resultingfusion protein preferably retains a site for a processing enzyme (factorXa) to cleave the bacteriophage protein from the MCP-1R gene or fragmentthereof (see Nagai, Nature 309:810(1984). Fusion proteins can also bemade with sequences from the lacZ gene (Jia, Gene 60:197(1987),the trpEgene (Allen, J. Biotechnol. 5:93(1987) and Makoff, J. Gen. Microbiol.135:11(1989), and the Chey gene (EP Patent Pub. No. 324 647) genes. TheDNA sequence at the junction of the two amino acid sequences may or maynot encode a cleavable site. Another example is a ubiquitin fusionprotein. Such a fusion protein is made with the ubiquitin region thatpreferably retains a site for a processing enzyme (e.g., ubiquitinspecific processing-protease) to cleave the ubiquitin from the MCP-1Rpolypeptide. Through this method, mature MCP-1R polypeptides can beisolated. See Miller, Bio/Technology 7:698(1989).

Alternatively, MCP-1R polypeptides can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of theMCP-1R polypeptides in bacteria. (See, for example, U.S. Pat. No.4,336,336). The signal sequence fragment typically encodes a signalpeptide comprised of hydrophobic amino acids which direct the secretionof the protein from the cell. The protein is either secreted into thegrowth media (gram-positive bacteria) or into the piroplasmic specie,located between the inner and outer membrane of the cell (gram-negativebacteria). Preferably there are processing sites, which can be cleavedeither in vivo or in vitro encoded between the signal peptide fragmentand the MCP-1R polypeptide.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) (Masui, Experimental Manipulation of Gene Expression (1983)and Ghrayeb, EMBO J. 3:2437(1984)) and the E. coli alkaline phosphatasesignal sequence (phoA) (see Oka, Proc. Natl. Acad. Sci. 82:7212(1985).The signal sequence of the alpha-amylase gene from various Bacilusstrains can be used to secrete heterologous proteins from B. subtilis(see Palva, Proc. Nati. Acad. Sci. 79:5582(1982) and EP Patent Pub. No.244 042).

Transcription termination sequences recognized by bacteria areregulatory regions located 3' to the translation stop codon. Togetherwith the promoter they flank the coding sequence. These sequences directthe transcription of an mRNA which can be translated into the MCP-1Rpolypeptide encoded by the MCP-1R DNA sequence (SEQ ID NOS:1 and 3).Transcription termination sequences frequently include DNA sequences ofabout 50 nucleotides capable of forming stem loop structures that aid interminating transcription. Examples include transcription terminationsequences derived from genes with strong promoters, such as the trp genein E. coli as well as other biosynthetic genes.

Typically, the promoter, signal sequence (if desired), coding sequenceof interest, and transcription termination sequence are maintained in anextrachromosomal element (e.g., a plasmid) capable of stable maintenancein the bacterial host. The plasmid will have a replication system, thusallowing it to be maintained in the bacterial host either for expressionor for cloning and amplification. In addition, the plasmid can be eithera high or low copy number plasmid. A high copy number plasmid willgenerally have a copy number ranging from about 5 to about 200, andtypically about 10 to about 150. A host containing a high copy numberplasmid will preferably contain at least about 10, and more preferablyat least about 20 plasmids.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectorstypically contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebacterial chromosome. See e.g., EP Patent Pub. No. 127 328.

Typically, extrachromosomal and integrating expression constructs cancontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and can include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline (see Davies, Ann. Rev. Microbiol.32:469(1978). Selectable markers can also include biosynthetic genes,such as those in the histidine, tryptophan, and leucine biosyntheticpathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are typicallycomprised of a selectable marker that is either maintained in anextrachromosal vector or an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal orintegrating, have been developed for transformation into many bacteria.Exemplary are the expression vectors disclosed in Palva, Proc. Natl.Acad. Sci. 79:5582(1982), EP Patent Pub. Nos. 036 259 and 063 953 andPCT Patent Publication WO 84/04541 (for B. subtilis); in Shimatake,Nature 292:128(1981), Amann, Gene 40:183(1985), Studier, J. Mol. Biol.189:113(1986) and EP Patent Pub. Nos. 036 776, 136 829 and 136 907 (forE.coli); in Powell, Appl. Environ. Microbiol. 54:655(1988) and U.S. Pat.No. 4,745,056 (for Streptococcus).

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and typically include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Exemplary methodologies can be found in Masson, FEMS Microbiol. Let.60:273(1989), Palva, Proc. Natl. Acad. Sci. 79:5582(1982), EP PatentPub. Nos. 036 259 and 063 953 and PCT Patent Pub. WO 84/04541 forBacillus transformation. For campylobacter transformation, see e.g.,Miller, Proc. Natl. Acad. Sci. 85:856(1988) and Wang, J. Bacteriol.172:949(1990). For E.coli, see e.g., Cohen, Proc. Natl. Acad. Sci.69:2110(1973), Dower, Nuc. Acids Res. 16:6127(1988), Kushner, GeneticEngineering: Proceedings of the International Symposium on GeneticEngineering (eds. H. W. Boyer and S. Nicosia), Mandel, J. Mol. Biol.53:159(1970) and Taketo, Biochem. Biophys. Acta 949:318(1988). ForLactobacillus and Pseudomonas, see e.g., Chassy, FEMS Microbiol. Let.44:173(1987) and Fiedler, Anal. Biochem. 170:38(1988), respectively. ForStreptococcus, see e.g., Augustin, FEMS Microbiol. Let. 66:203(1990),Barany, J. Bacteriol. 144:698(1980), Harlander, Streytococcal Genetics(ed. J. Ferretti and R. Curtiss III)(1987), Perry, Infec. Immun.32:1295(1981), Powell, Appl. Environ. Microbiol. 54:655(1988) andSomkuti, Proc. 4th Evr. Cong. Biotechnology 1:412(1987).

III. Expression and Detection of Expressed MCP-1R Proteins

In order to obtain MCP-1R expression, recombinant host cells derivedfrom the transformants are incubated under conditions which allowexpression of the MCP-1R encoding sequence (SEQ ID NOS:1 AND 3). Theseconditions will vary, depending upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

Detection of an MCP-1R protein expressed in the transformed host cellcan be accomplished by several methods. For example, detection can be byenzymatic activity (or increased enzymatic activity or increasedlongevity of enzymatic activity) using fluorogenic substrates which arecomprised of a dibasic cleavage site for which an MCP-1R protein isspecific. An MCP-1R protein can also be detected by its immunologicalreactivity with anti-MCP-1R antibodies.

IV. Identification of MCP-1 Receptor Antagonists

Different ligands of a cellular receptor are classified on the basis oftheir capacity to induce biological responses. Substances that are bothcapable of binding to the receptor and triggering a response areclassified as agonists. By contrast, ligands that are capable of bindingto the receptor but are incapable of triggering a response areclassified as antagonists. Antagonists compete, sometimes extremelyeffectively, with the natural ligand or its agonists, leading tofunctional receptor inactivation (receptor antagonism).

A method is provided for identifying ligands of the MCP-1 receptor, suchas antagonists. The method comprises transfecting a mammalian cell linewith an expression vector comprising nucleic acid sequences encoding theN-terminal domain of MCP-1 receptor (see SEQ ID NOS:1 and 3). TheN-terminal domain of the MCP-1 receptor may be expressed alone or incombination with other domains of the MCP-1 receptor. The other domainsmay be extracellular, intracellular or transmembrane domains. Moreover,a chimaeric protein may be expressed, where the other domains are thecorresponding domains from related proteins, such as those in FIG. 4(SEQ ID NOS:5, 6, 7 and 8). The N-terminal domain may also be expressedas a portion of the native MCP-1 receptor. Expression of extracellulardomains is preferred where soluble protein for solid phase assays isrequired.

The antagonist is identified by adding an effective amount of an organiccompound to the culture medium used to propogate the cells expressingthe N-terminal domain of MCP-1 receptor. An effective amount is aconcentration sufficient to block the binding of MCP-1 to the receptordomain. The loss in binding of MCP-1 to the receptor may be assayedusing various techniques, using intact cells or in solid-phase assays.

For example, binding assays similar to those described for IL-7 in U.S.Pat. No. 5,194,375 may be used. This type of assay would involvelabelling MCP-1 and quantifying the amount of label bound by MCP-1receptors in the presence and absence of the compound being tested. Thelabel used may, for example, be a radiolabel, e.g. ¹²⁵ I or afluorogenic label.

Alternatively, an immunoassay may be employed to detect MCP-1 binding toits receptor by detecting the immunological reactivity of MCP-1 withanti-MCP-1 antibodies in the presence and absence of the compound beingtested. The immunoassay may, for example, involve an antibody sandwichassay or an enzyme-linked immunoassay. Such methods are well known inthe art and are described in Methods in Enzymology, Volumes 154 and 155(Wu and Grossman, and Wu, Eds., respectively), (Mayer and Walker, Eds.)(1987); Immunochemical Methods in Cell and Molecular Biology (AcademicPress, London).

Pharmaceutical compositions comprising the MCP-1 receptor antagonist maybe used for the treatment of disease characterized by monocyticinfiltrates, such as rheumatoid arthritis and alvcolitis. The antagonistis administered as a pharmaceutical composition comprising atherapeutically effective amount of the antagonist and apharmaceutically acceptable vehicle. Such pharmaceutical compositionsmay also contain pharmaceutically acceptable carriers, diluents,fillers, salts, buffers, stabilizers and/or other materials well-knownin the art. The term "pharmaceutically acceptable" means a material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s) and that is not toxic to the host to which itis administered. The characteristics of the carrier or other materialwill depend on the route of administration.

Administration can be carried out in a variety of conventional ways.Parenteral administration is currently preferred. In such cases, theantagonist composition may be in the form of a non-pyrogenic, sterile,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable solutions, having due regard to pH, isotonicity,stability and the like, is within the skill in the art. In the longterm, however, oral administration will be advantageous, since it isexpected that the active antagonist compositions will be used over along time period to treat chronic conditions.

The amount of active ingredient will depend upon the severity of thecondition, the route of administration, the activity of the antagonist,and ultimately will be decided by the attending physician. It iscurrently contemplated, however, that the various pharmaceuticalcompositions should contain about 10 micrograms to about 1 milligram permilliliter of antagonist.

In practicing the method of treatment of this invention, atherapeutically effective amount of the antagonist composition isadministered to a human patient in need of such treatment as a result ofhaving a condition characterized by monocytic infiltrates. The term"therapeutically effective amount" means the total amount of the activecomponent of the method or composition that is sufficient to show ameaningful patient benefit, i.e., healing of chronic conditions orincrease in rate of healing. A therapeutically effective dose of anantagonist composition of this invention is contemplated to be in therange of about 10 micrograms to about 1 milligram per milliliter perdose administered. The number of doses administered may vary, dependingon the individual patient and the severity of the condition.

The invention is further described in the following examples, which areintended to illustrate the invention without limiting its scope.

V. Examples

Standard procedures for the isolation and manipulation of DNA are fromSambrook, et al. (1989). Plasmid DNA was propagated in E. coli strainsHB101, D1210 or XL-1 Blue (Stratagene). DNA sequencing was performed bythe dideoxy chain termination method (Sanger, 1977) using M13 primers aswell as specific internal primers.

EXAMPLE 1 PCR Identification of cDNA Clones

To identify and clone new members of the chemokine receptor gene family,degenerate oligonucleotide primers were designed and synthesizedcorresponding to the conserved sequences NLAISDL (SEQ ID NO: 11) in thesecond and DRYLAIV (SEQ ID NO:12) in the third transmembrane domains ofthe MIP-1α/RANTES receptor, the IL-8 receptors and the HUMSTSR orphanreceptor (GenBank Accession #M99293). Amplification of cDNA derived fromMM6 cells with the primers yielded a number of PCR productscorresponding in size to those expected for a seven transmembranereceptor. Analysis of the subcloned PCR products revealed cDNAs encodingthe predicted fragments of the receptors from which the primers weredesigned as well as one cDNA that appeared to encode a novel protein. Toobtain a full-length version of this clone, a MM6 cDNA library wasconstructed in pFROG and probed by hybridization with the PCR product. A2.1 kb cDNA clone was obtained. Analysis of additional clones in the MM6cDNA library revealed a second sequence that was identical to the 2.1 kbcDNA sequence first obtained from the 5' untranslated region through theputative seventh transmembrane domain but contained a differentcytoplasmic tail from the 2.1 kb cDNA sequence first obtained. Twoindependent clones in the library were found to contain the secondsequence, which appears to represent alternative splicing of thecarboxyl-terminal tail of the MCP-1R protein. The two sequences aredenoted MCP-1RA and MCP-1RB, monocyte chemoattractant protein-1receptors A and B, representing, respectively, the first and secondsequences isolated (SEQ ID NOS:1, 2, 3 and 4). Details of the materialsand methods used follow.

1. Oligonucleotide Synthesis

Oligonucleotide adapters, probes, and primers were synthesized on anApplied Biosystems (Foster City, Calif.) instrument according to themanufacturer's instructions. The degenerate oligonucleotide primerscorresponding to conserved sequences in the second and thirdtransmembrane domains as noted above and incorporating EcoRI and XhoIrestriction sites in their 5' ends that were used to identify MCP-1Rwere a 27-mer, 5' CGC TCG AGA CCT (G or A)(G or T)C (C or A)(A, T or G)T(T or G)(T or G)C (T or C)GA CCT 3' (SEQ ID NO:9) and a 31-mer 5' GC GAATTC TGG AC(G or A) ATG GCC AGG TA(C,A or G) C(T or G)G TC 3' (SEQ IDNO:10).

2. Polymerase Chain Reactions (PCR)

MM6 cells, which are derived from a human monocytic leukemia (see Weber,Eur. J. Immunol. 23:852-59(1993)) were obtained from the DSM GermanCollection of Microorganisms and Cell Cultures, Masheroder Weglb, 3300Braunschweig, Germany. The cells were grown in RPMI-1640 (GIBCO BRL,Grand Island, N.Y.), supplemented with 10% fetal calf serum, 25 mMHepes, and antibiotics. Total RNA was isolated from the MM6 cells by themethod of Chomczynski and Sacchi. See Chomczynski, Anal. Biochem.162:156-59(1987). Poly A⁺ RNA was obtained by affinity chromatography onoligo dT cellulose columns (Pharmacia, Piscataway, N.J.). First strandcDNA synthesis was performed starting with 5 μg of MM6 poly A+ RNAaccording to the manufacturer's instructions (Pharmacia).

PCR reactions were carried out for 30 cycles beginning with a 1-minuteincubation at 94° C., 2 minutes at 50° C., 1.5 minutes at 72° C., and afinal elongation step at 72° C. for 4 minutes using the PCR primersdescribed above (SEQ ID NOS:9 and 10) at a final concentration of 1 μMand MM6 cDNA at approximately 10 ng/ml. PCR products migrating between200-300 base pairs on a 1.5% agarose gel were excised, subcloned intopBluescript (sk⁻) and sequenced using fluorescently labeleddideoxyribonucleotides as described by Sanger, Proc Natl Acad SciUSA74:5463-67(1977). Sequence analysis revealed cDNAs encoding thepredicted fragments of the receptors upon which the primers weredesigned and one cDNA which appeared to encode a novel protein. Toobtain a full-length version of this clone, an appropriate cell line waschosen and a cDNA library was constructed in pFROG and probed with thisPCR product, as detailed in subsections 3 and 4 below.

3. Identification of the MM6 Cell Line

Because monocytes are difficult to isolate in usable quantity andexpress less than 2000 high affinity MCP-1 binding sites per cell, acultured cell line that responded well to MCP-1 had to be identified.Using the calcium efflux assay as described in Vu, Cell64:1057-68(1991), MCP-1 induced calcium fluxes in various cell lineswere measured. No calcium flux was detected in undifferentiated humanHL-60 cells and human erythroleukemia (HEL) cells. In contrast, adose-dependent calcium flux was detected in MM6 cells, with half maximalstimulation at 4 nM MCP-1. The response of MM6 cells to MCP-1 could notbe ablated by prior exposure to RANTES, whereas the response to RANTESwas partially blocked by prior exposure to MCP-1. Similar resultsobtained when MIP-1α was used instead of RANTES.

4. Expression Cloning of MCP-1 Receptor

The overall strategy for cloning the MCP-1 receptor was to confer MCP-1responsiveness to Xenopus oocytes that were microinjected with RNAencoding the receptor. This methodology has been successfully employedto clone the 5-HT, thrombin, IL8RA, and MIP-1α/RANTES receptors. Oocytesare harvested from gravid frogs, and treated with collagenase to removethe follicular cells. The cDNA library is electroporated into bacterialhost cells which are then divided into pools of 5,000 to 50,000colonies/petri dish. DNA is prepared from each pool of bacteria andlinearized. One day after harvesting, the oocytes are microinjected withpoly A+ RNA or cRNA transcribed from the linearized cDNAs and incubatedfor two days to allow protein expression. On the day of the experiment,the oocytes are loaded with ⁴⁵ Ca, washed to remove unincorporated ⁴⁵Ca, and then incubated with potential ligands. In the presence of theappropriate ligand a significant afflux of ⁴⁵ Ca is detected. Uninjectedoocytes are used as controls. A minimum of 1,000,000 colonies arescreened (i.e., 20 to 200 pools) and if a positive pool is found it issubdivided (sibed) into smaller pools which are then individuallyscreened. The process is repeated until a single clone is obtained.

As a prelude to undertaking this very labor intensive approach, poly A+RNA from large scale preparations of THP-1 and MM6 cells was injectedinto oocytes, but failed to confer MCP-1 dependent signaling.Furthermore, larger mRNA species were enriched by size fractionation of200-300 μg of poly A+ THP-1 and MM6 RNA on sucrose gradients beforeinjecting individual fractions into oocytes. Once again MCP-1 dependentsignaling in oocytes was not demonstrated. In addition, injection of alimited number of cRNAs transcribed from library pools also failed toconfer signaling. These experiments suggested that the MCP-1 receptormessage is most likely of low abundance, and not detectable in a poolsize large enough to make expression cloning by sib-selection feasible.For this reason, the polymerase chain reaction (PCR)-based strategy waspursued.

5. Construction and Screening of the MM6 cDNA Library

A cDNA library was constructed in the vector pFROG, a modified versionof pCDM6 that includes approximately 100 bases of 5' untranslatedxenopus globin sequence just 3' of the SP6 promoter, as described by Vu,Cell 64:1057-68 (1991).

After first strand and second strand synthesis from MM6 poly A⁺ RNA wasperformed (see subsection 2 above), the cDNA was size selected for 2 kbor greater by agarose gel electrophoresis. BstXI linkers were added forinsertion into the pFROG vector. After ligation, pFROG waselectroporated into competent MC1061p3 cells. A total of 1,000,000colonies were screened by hybridization under conditions of highstringency (50% foramide, 6×SSC, 0.1% SDS, 42° C., 16h) as described inSambrook, Molecular Cloning: A Laboratory Manual, Second Edition (1989)using the novel PCR product isolated as described in subsection 2 above.Positives were sequenced using fluorescently labeleddideoxyribonucleotides as described by Sanger, Proc. Natl. Acad. Sci.74:5463 (1977). Two cDNA clones containing the A form of the receptorand two clones containing the B form were isolated.

EXAMPLE 2 Structure of MCP-1R Deduced from the cDNA Sequence

The full sequence of MCP-1RA cDNA (SEQ ID NO:1) and the encoded aminoacid sequence (SEQ ID NO:2) are shown in FIGS. 1A-1D. The encodedprotein sequence is shown below that of the cDNA sequence. The cDNAsequence (SEQ ID NO:3) and encoded amino acid sequence (SEQ ID NO:4) ofMCP-1RB are shown in FIGS. 2A-2C. Conventional numbering is used.

The translation of both MCP-1R DNAs is most likely initiated at the ATGstart codon. This is the only in-frame MET codon in the 5' region of thecDNA. Following the initiating methionine (MET) is an open reading frameencoding a protein of 374 amino acids with a predicted molecular weightof about 42,000 Daltons. By direct comparison with the knowntransmembrane domains for the MIP-1α/RANTES receptor, the orphanreceptor HUMSTSR and the IL-8 receptors 8RA and 8RB, an extra cellularamino terminus of 48 residues is revealed. The transmembrane domains aremost likely located at amino acids 49 through 70, 80 through 700, 115through 136, 154 through 178, 204 through 231, 244 through 268 and 295through 313. They are indicated in FIG. 4 by the horizonal lines abovethe sequence groupings (SEQ ID NOS: 2, 5, 6, 7 and 8). The carboxyl tailof 61 amino acids begins with serine at position 314 (see FIGS. 4A-4B).

The MCP-1RB cDNA encodes an amino acid sequence identical to that ofMCP-1RA from the MET at position 1 through the arginine at position 313and including 30 untranslated nucleotides immediately 5' of theinitiating MET (see FIGS. 2A-2C). The putative amino acid sequence ofMCP-1RB (SEQ ID NO:4) however reveals a completely different cytoplasmictail than the 61 amino acid cytoplasmic tail of MCP-1RA (SEQ ID NO:2).MCP-1RB has a cytoplasmic tail of 47 amino acids beginning with arginineat amino acid position 314 and ending with leucine at position 360. Thatalternative splicing occurred at position 313 can be inferred from thesequence identity, including the 5' untranslated sequence, of the twoclones and from the characteristic AG sequence located at the putativedonor junction between amino acid positions 313-314. In addition, a cDNAcommon to both A and B forms of MCP-1R hybridized to a single band onSouthern blots of human genomic DNA under high stringency conditions,and one cDNA clone from the MM6 library was obtained that contained intandem both carboxyl-terminal cytoplasmic tails found in MCP-1RA and1RB, suggesting derivation from incompletely processed RNA. The MCP-1receptor, MCP-1R, is only the second known example of alternativesplicing of the carboxyl tails of receptors in the seven-transmembranereceptor family. Namba, Nature 365:166-70(1993) has reported that theprostaglandin (PG) E2 receptor has four alternatively splicedcarboxyl-terminal tails with little sequence homology among the four.The related MIP-1α/RANTES and IL-8 receptors are believed to beintronless. See Holmes, Science 253:1278-80(1991); Murphy, Science253:1280-83(1991) and Neote, Cell 72:415-25(1993). Alignment of thecytoplasmic tails of MCP-1RA and 1RB with other chemokine receptorsrevealed that one of the receptors, MCP-1RB, was homologous to thecorresponding region in the MIP-1α/RANTES receptor. The carboxyl tail ofMCP-1RA bore no significant identity with other known proteins.

Northern blots of hematopoietic cell lines were performed as describedin Sambrook, Molecular Cloning: A Laboratory Manual, Second Edition(1989), and probed for each of the MCP-1R clones revealed that both mRNAspecies migrated as a single 3.5 kb band. See FIGS. 3A-3B. Both mRNAswere expressed at approximately equal levels in the MCP-1 responsivecell lines MM6 and in THP-1 cells. Neither were expressed in theunresponsive cell lines HEL and HL-60. Expression of each of the mRNAwas also detected in freshly isolated human monocytes by reversetranscription PCR.

EXAMPLE 3 Similarity of MCP-1RA and 1RB to Other Seven MemberTransmembrane Receptors

Comparison of the sequences of MCP-1RA (SEQ ID NO:2) with the IL-8receptors RA and RB, the MIP-1α/RANTES receptor and the orphan receptorHUMSTSR (SEQ ID NOS:7, 8, 5 and 6, respectively) is illustrated in FIGS.4A-4B. Comparison of the deduced amino acid sequence of the novel MCP-1Areceptor with other seven transmembrane proteins revealed that it mostclosely relates to the MIP-1α/RANTES receptor, with 51% identity at theamino acid level. The IL-8 receptors RA and RB exhibited 30% identity atthe amino acid level to and the HUMSTSR orphan receptor exhibited 31%identity at the amino acid level. Analysis reveals that the MCP-1receptor has diverged from the related MIP-1α/RANTES receptor and theIL-8 receptors in its amino-terminal and carboxyl-terminal domains. Astriking identity between the MCP-1A receptor and the MIP-1α/RANTESreceptor is found in the sequence IFFHLLTI DRYLAIV HAVFAL(K/R) ARTVTFGV(SEQ ID NOS: 13 and 14), which occurs at the end of the thirdtransmembrane domain (see FIGS. 4A-4B). The corresponding region ofrhodopsin is known to participate in G-protein binding (Franke et al.,Science 250:123 (1990)), suggesting that this domain may mediate aspectsof G-protein activation common to receptors for C-C chemokines.

EXAMPLE 4 Confirmation of Receptor Activity

The calcium efflux assay was performed to confirm expression offunctional MCP-1R protein and to determine whether the MCP-1 receptors Aand B conferred responsiveness to MCP-1 or other chemokines. In thisassay, MCP-1RA and 1RB cRNA was microinjected into Xenopus oocytes andreceptor signaling activities measured by detection of agonist-inducedcalcium mobilization. Signaling activity by the MIP-1α/RANTES receptorand the IL-8 receptor RA was examined in parallel.

As described in Vu, Cell 64:1057-68(1991), cRNA was prepared by SP6 RNApolymerase transcription from a NotI linearized vector and run on anagarose gel to confirm a single band of the expected size. One day afterharvesting, oocytes were injected with 20 ng of cRNA in a total volumeof 50 nl per oocyte. After incubation in modified Barth's buffer for 2days at 16° C., the oocytes were loaded with Ca⁴⁵ (50 uCi/ml, Amersham,Arlington Heights, Virginia) for 3 hours, washed for one hour, andplaced into wells of a 24-well dish in groups of seven, in a volume of0.5 ml Ca⁴⁵ efflux was determined by collecting the media at 10 minuteintervals and counting beta emissions in a liquid scintillation counter.After a stable baseline had been achieved, cytokine agonists MIP-1α,MIP-1β, RANTES, IL-8 and MCP-1 were added in the Barth's media to theoocytes for 10 minutes. Uninjected oocytes were used as controls. Thecytokines, MIP-1α, MIP-1β, RANTES, IL-8 and MCP-1 were obtained from R&DInc., Minneapolis, Minn. The results are shown in FIG. 6.

Both MCP-1RA and 1RB conferred robust and remarkably specific responsesto nanomolar concentrations of MCP-1. No response was elicited by thechemokines MIP-1α, MIP-1β, RANTES, or IL-8, even when these ligands werepresent at 500 nM. In contrast, the MIP-1α/RANTES receptor signaled inresponse to MIP-1α and RANTES, but not to MCP-1, consistent withpublished results. The EC₅₀ for MCP-1 was 15 nM.

EXAMPLE 5 MCP-1R Ligand Specificity and Signal Transduction

A. Ligand Specificity

A cell line stably expressing an MCP-1R receptor was produced bytransfection of MCP-1RB cDNA into HEK-293 cells.

Human embryo kidney (HEK)-293 (CRL 1573) cells were obtained from theAmerican Type Culture Collection (Bethesda, Md.) and were grown inMinimal Essential Media with Earle's Balanced Salt Solution(MEM-EBSS;GIBCO/BRL, Grand Island, N.Y.) supplemented with 10% fetalcalf serum ("FCS") (Hyclone Laboratories Inc., Logan, Utah) and 1%penicillin/streptomycin, at 37° C. in a humidified 5% CO₂ atmosphere.cDNAs for the MCP-1 receptor, MCP-1RB, and the MIP-1α/RANTES receptorwere cloned into the polylinker of the mammalian cell expression vectorpcDNA3 (Invitrogen Inc., San Diego, Calif.) and transfected into 293cells (50-80% confluent) with a DNA/Lipofectamine (GIBCO/BRL) mixtureaccording to the manufacturer's instructions. After selection for 2-3weeks in the presence of G418 (0.8 mg/ml) (GIBCO/BRL), colonies werepicked and stable cell lines were screened by northern blot analysis forreceptor expression. In general, there was a strong correlation betweenthe level of receptor expression as judged by northern blot analysis andthe strength of the receptor signals obtained in the below describedfunctional assays. Transfected cells that failed to express the receptoron northern blots were used as negative controls in the binding andsignaling experiments.

Equilibrium binding assays were then performed using the method ofErnst, J. Immunol. 152: 3541-49 (1994). Varying amounts of ¹²⁵ I-labeledMCP-1 (Dupont-NEN, Boston, Mass.) were incubated with 6×10⁶ MCP-1RBexpressing HEK-293 cells resuspended in binding buffer (50 mM Hepes, pH7.2, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA (bovine serum albumin, fraction V,Sigma)) in the presence or absence of 100-fold excess of the unlabeledC-C chemokines MIP-1α, MIP-1β and RANTES, and the C-X-C chemokine IL-8(chemokines obtained from R&D Systems, Inc., Minneapolis, Minn).Competition experiments were performed using 500 pM ¹²⁵ I-labeled MCP-1and the concentrations of unlabeled chemokines as indicated in FIGS.7A-7B.

Equilibrium binding data were analyzed according to the method ofScatchard using the program "LIGAND" (Biosoft, Ferguson, Mo.) on aMacintosh computer. See Munson, Anal. Biochem. 107: 220-39 (1980). Theclosely related C-C chemokines MIP-1α, MIP-1β, and RANTES, as well asthe C-X-C chemokine IL-8 did not compete for binding. Nor was specificbinding detected in transfectants that expressed little or no MCP-1RB onNorthern blots. Analysis of equilibrium binding data shown in FIG. 7indicates a dissociation constant (K_(d)) of 260 pM (FIG. 7B). ThisK_(d) is in good agreement with that reported for the binding of MCP-1to monocytes (Yoshimura, J. Immunol. 145:292-97 (1990); Zhang, J. Biol.Chem. 269:15918-24 (1994)) and THP-1 cells (Van Riper, J. Exp. Med.177:851-56 (1933)). These data indicate that ¹²⁵ I-MCP-1 boundspecifically and with high affinity to the MCP-1RB receptor expressed in293 cells.

B. Sienal Transduction

Calcium mobilization in 293 cells was then investigated. TransfectedHEK-293 cells were grown until confluent, trypsinized briefly, washedwith phosphate buffered saline containing 1 mg/ml BSA (PBS-BSA), andresuspended in serum-free MEM-EBSS supplemented with 1 mg/ml BSA and 10mM HEPES (pH 7.0) at a density of 2×10⁷ cells/ml. The cells wereincubated in the dark at 37° C. for 20 min in the presence of 5-10 μg/mlindo-1 AM (Molecular Probes, Inc., Eugene, Oreg.). Nine volumes ofPGS-BSA were added, and the cells were incubated for an additional 10min at 37° C., pelleted by centrifugation, and washed twice with 50 mlof the PBS-BSA solution. Washed, indo-1-loaded cells were thenresuspended in Hank's Balanced Salt Solution (1.3 mM Ca²⁺) supplementedwith 1 mg/ml BSA (HBS-BSA) at a density of approximately 0.5×10⁶cells/ml at room temperature. To measure intracellular calcium ([Ca²⁺]_(i)), 0.5 ml of the cell suspension was placed in a quartz cuvette ina Hitachi F-2000 fluorescence spectrophotometer. Chemokines (MCP-1,RANTES, MIP-1α, MIP-1β, Gro-α and IL-8) dissolved in HBS-BSA wereinjected directly into the cuvette in 5 μl volumes. Intracellularcalcium was measured by excitation at 350 nm and fluorescence emissiondetection at 490 nm (F1) and 410 nm (F2) wavelengths. The [Ca²⁺ ]_(i)was estimated by comparing the 490/410 fluorescence ratio after agonistapplication (R) to that of calibration ratios measured at the end ofeach run, according to the equation:

    [Ca.sup.2+ ].sub.i =K.sub.d ×[(R-R.sub.min)/(R.sub.max -R)]×(Sf2/Sb2)

where R_(max) and R_(min) represent the fluorescence ratio undersaturating (1.3 mM Ca²⁺) and nominally free (10 mM EGTA, Sigma ChemicalCo.) calcium conditions, K_(d) is the dissociation constant of calciumfor indo-1, R is the fluorescence ratio, and Sf2/Sb2 is the fluorescenceratio of free and bound indo-1 dye at 410 nm. See Thomas, A P andDelaville, F (1991) in Cellular Calcium, a Practical Approach, OxfordUniv. Press, pp. 1-54.

To quantitate calcium responses, MCP-1 dose response curves weregenerated in each experiment and the results were expressed as a percentof the maximum calcium signal (at 300 nM MCP-1) measured in thatexperiment. The changes in [Ca²⁺ ]_(i) levels in response to eachconcentration of agonist were determined by subtracting the baselinefrom peak [Ca²⁺ ]_(i) levels, which were determined by averaging 5seconds of data prior to agonist addition and surrounding the peakresponse, respectively. In experiments done to determine the role ofextracellular calcium, 3 mM EGTA was added 60-90 seconds prior to MCP-1.Subsequent lysis of the cells with Triton X-100 (Sigma) caused no changein indo-1 fluorescence, indicating that EGTA had reduced theextracellular calcium concentration below that of intracellular basallevels (approximately 70-100 nM). All experiments were performed at roomtemperature.

MCP-1 stimulated robust calcium mobilization in the stably transfectedMCP-1RB/293 cells in a specific and dose-dependent manner. Small butreproducible signals were seen with as little as 100 pM MCP-1, and theaverage EC₅₀ from four full dose-response curves to MCP-1 was 3.4 nM(2.7-4.4 nM; FIGS. 8A and 8B). The MCP-1RB receptor was selectivelyactivated by MCP-1. RANTES, MIP-1α, MIP-1β, Gro-α, and IL-8 failed tostimulate significant calcium signals in these same cells, even whenpresent at high concentrations (FIG. 8B). Furthermore, these chemokinesalso failed to block stimulation of the cells by MCP-1, indicating thatthey are unlikely to act as endogenous antagonists of the MCP-1RBreceptor. The MCP-1-dependent intracellular calcium fluxes werecharacterized by short lag times, followed by a rapid rise in [Ca²⁺]_(i) that returned to near basal levels within 80-90 sec of theaddition of MCP-1 (FIG. 8A). The cells demonstrated homologousdesensitization in that they were refractory to activation by a secondchallenge with MCP-1 (FIG. 8C).

To determine the source of the intracellular calcium flux, theMCP-1RB/293 cells were challenged with MCP-1 in the presence or absenceof extracellular calcium. The rise in cytoplasmic calcium was largelyunchanged by the chelation of extracellular calcium with 3 mM EGTA.Similar results were seen when the cells were washed and resuspended incalcium-free PBS supplemented with 1 mM EGTA, or when 5 mM Ni²⁺ wasadded to the cuvette to block the influx of extracellular calcium.Sozani, J. Immunol. 147:2215-21 (1991); Saga, J. Biol. Chem.262:16364-69 (1987). The fall in cytoplasmic calcium to baseline wasslightly prolonged in the presence of extracellular calcium, suggestingthat calcium influx may contribute to maintaining the response to MCP-1after intracellular stores are depleted. These data suggest that theprimary means of calcium mobilization in these transfected 293 cells isthrough release of intracellular calcium.

Inositol (1,4,5)-triphosphate (IP₃) mobilizes intracellular calcium inresponse to activation of a wide spectrum of receptors, including manyseven-transmembrane-domain receptors. Hung, J. Cell. Biol. 116:827-32(1992); Putney, Trends Endocrinol. Metab. 5:256-60 (1994). Toinvestigate this mobilization, total inositol phosphate accumulation wasdetermined as described in Hung, J. Cell Biol. 116: 827-32 (1992).HEK-293 cells were grown until confluent in 24-well tissue culturedishes and labeled overnight with 2 uCi/ml [³ H]myo-inositol (23Ci/mmol) (New England Nuclear, Boston, Mass.) in inositol-free MEM-EBSSsupplemented with 10% dialyzed FCS. Following labeling, the media wereremoved and the cells were incubated at room temperature for 5-10 min in0.5 ml of serum-free MEM-EBSS media supplemented with 10 mM HEPES, 1mg/ml BSA, and 10 mM LiCl. The washed cells were then incubated with thechemokines MCP-1, MIP-1α, MIP-1β, RANTES, IL-8 and Gro-α for 1-30 min atroom temperature in the presence of 10 mM LiCl. The incubation wasterminated by removal of the incubation media and addition of 1 ml ofice-cold 20 mM formic acid. Plates were incubated at 4° C. for 30 minbefore the supernatants were applied to 1-ml Dowex AG1-X8 (100-200 mesh,formate form, from Sigma) chromatography columns. Columns were washedwith 8 ml of water followed by 5 ml of 40 mM sodium formate. Total [³H]inositol phosphates were eluted with 5 ml of 2 M ammonium formate/0.1M formic acid and quantitated by liquid scintillation spectroscopy.Activation of the MCP-1 receptor in transfected 293 cells induced littleor no hydrolysis of phosphatidyl inositol. In control experimentsactivation of the muscarinic (Lameh, J. Biol. Chem. 267:13406-412(1992)) or oxytocin receptor, Kimura, Nature 356:526-29 (1992),co-transfected into these same 293 cells, led to a 5- to 9-fold increasein PI turnover.

To investigate inhibition of adenylyl cyclase activity, HEK-293 cellsstably-transfected with the MCP-1RB receptor and the MIP-1α/RANTESreceptor were grown until confluent in 24-well tissue culture dishes andlabeled overnight with 2 μCi/ml of [³ H]adenine (25-30 Ci/mmol) (NewEngland Nuclear, Boston, Mass.) in MEM-EBSS supplemented with 10% FCS.The next day, the cells were washed by incubation at room temperaturewith 0.5 ml of serum-free MEM-EBSS media supplemented with 10 mM HEPES,1 mg/ml BSA, and 1 mM IBMX (3-isobutyl-1-methylxanthine) for 5 min.After removal of the wash media the cells were stimulated by addition offresh media containing either chemokine (MCP-1, MIP-1α, MIP-1β, RANTES,IL-8 and Gro-α) alone, forskolin alone (10 μM, Sigma Chemical Co., St.Louis, Mo.), or chemokine plus forskolin, all in the presence of 1 mMIBMX, for 20 min at room temperature. The incubation was terminated byreplacement of the media with 1 ml of ice-cold 5% TCA (trichloroaceticacid), 1 mM cAMP, and 1 mM ATP (Sigma). Following incubation at 4° C.for 30 min, the labeled [³ H]ATP and [3H]cAMP pools were separated andquantitated by chromatography on Dowex 50W (200-400 mesh, hydrogen form,from Sigma) and neutral alumina columns (also from Sigma), as describedin Hung, J. Biol. Chem. 267: 20831-34 (1992) and Wong, Nature 351: 63-65(1991). The 1 ml acid supernatant was loaded onto a 1-ml Dowex 50Wcolumn and the ATP pool eluted with 3 ml of H₂ O. The Dowex 50W columnswere then placed over 1-ml alumina columns, and 10 ml of H₂ O was addedto the Dowex resin and the eluant allowed to drop directly onto theneutral alumina. The cAMP pool was then eluted directly from the aluminawith 5 ml of 0.1 M imidazole/0.01 mM sodium azide. The [³ ]ATP and [³H]cAMP fractions were counted by liquid scintillation spectroscopy. ThecAMP pool for each sample was normalized to its own ATP pool andexpressed as a ratio by the equation (cAMP cpms/ATP cpms)×100. In eachexperiment full dose-response curves were generated and expressed as apercent of the forskolin control.

Activation of the MCP-1 receptor resulted in a potent and dose-dependentinhibition of adenylyl cyclase activity. MCP-1 significantly reducedbasal cAMP accumulation in these cells by 55% (p<0.01, Student's ttest). Forskolin activation of adenylyl cyclase increased cAMP levels16-fold, and co-addition of MCP-1 blocked this increase by 78%, with anIC₅₀ of 90 mM (70-140 pM). The magnitude and potency of MCP-1 inhibitionof adenylyl cyclase activity was independent of the forskolinconcentration (3-30 μM). MCP-1 neither stimulated nor inhibited cAMPformation in untransfected or pcDNA3 transfected 293 cell controls.

Together these results demonstrate that inhibition of adenylyl cyclaseactivity provides a sensitive and quantitative assay for MCP-1RBreceptor activation in 293 cells. Virtually no activation of the MCP-1receptor could be detected in this assay in response to highconcentrations of RANTES, MIP-1α, MIP-1β, IL-8, or Gro-α which isconsistent with our observations in the calcium fluorimetric assay andin Xenopus oocytes (Example 5).

In similar experiments the MIP-1α/RANTES receptor was stably transfectedinto 293 cells and also found to mediate potent and dose-dependentinhibition of adenylyl cyclase activity. Unlike the MCP-1RB receptor,however, the MIP-1α/RANTES receptor was activated by multiple chemokineswith varying degrees of potency. MIP-1α and RANTES were virtuallyequipotent in inhibiting adenylyl cyclase activity with IC₅₀ s of 110 pMand 140 pM, respectively. MIP-1β (IC₅₀ =820 nM) also inhibited adenylylcyclase activity, though only at much higher concentrations, and neitherblocked cAMP accumulation to the same extent as MIP-1α and RANTES. TheC-X-C chemokines IL-8 and Gro-α did not activate the MIP-1α/RANTESreceptor at up to 1 μM.

Table I below compares the activation of the MCP-1 receptor and theMIP-1α/RANTES receptor by a variety of chemokines and demonstrates thespecificity of the MCP-1RB receptor for MCP-1, and the MIP-1α/RANTESreceptor for MIP-1α and RANTES. Neither of the C-X-C chemokines wasactive on either of the two cloned C--C chemokine receptors.

                  TABLE I                                                         ______________________________________                                        Specificity of the MCP-1 and MIP-1α/RANTES Receptors                      Inhibition of Adenylyl Cyclase                                                               MIP-1α/                                                  MCP-1RB RANTES R                                                            IC.sub.50 (nM)     Selectivity                                                ______________________________________                                        MCP-1   .090     820       >9000 for MCP-1R                                     MIP-1α >10.sup.3 .110 >9000 for MIP-1α/RANTES R                   RANTES >10.sup.3 .140 >7000 for MIP-1α/RANTES R                         MIP-1β >10.sup.3 10  >100 for MIP-1α/RANTES R                      Gro-α >10.sup.3 >10.sup.3                                               IL-8 >10.sup.3 >10.sup.3                                                    ______________________________________                                    

In all experiments, the maximum inhibition of adenylyl cylase activitymediated by the MCP-1RB or MIP-1α/RANTES receptor was ˜80% and ˜55%,respectively. Qualitatively similar signaling, manifested by the rapidrise in cytoplasmic calcium and potent inhibition of adenylyl cyclase,was observed in 293 cells expressing the MCP-1RA receptor.

C. Inhibition of MCP-1R Activation

Inhibition of MCP-1RB receptor activation by bordetella pertussis toxinwas investigated. Pertussis toxin (List Biological Labs, Inc., Campbell,Calif.) was dissolved in 0.01 M sodium phosphate, pH 7.0, 0.05 M sodiumchloride and diluted into normal serum containing media at finalconcentrations of 0.1 ng/ml to 100 ng/ml, and incubated with cellsovernight (14-16 h) at 37° C. The conditions of the Pertussis toxintreatment of the 293 cells were identical for calcium fluorimetric andadenylyl cyclase experiments. In the adenylyl cyclase experiments, thePertussis toxin was added at the same time as [³ H]adenine.

The MCP-1-induced mobilization of intracellular calcium, as well as theinhibition of adenylyl cyclase, was substantially blocked bypretreatment of cells with bordetella pertussis toxin. Dose-responsestudies indicated a similar degree of inhibition of these two pathwaysby pertussis toxin, as well as a component (≈20%) that was resistant toinhibition by up to 100 ng/ml of PT. The effect of pertussis toxintreatment was to reduce the magnitude of the MCP-1 inhibition of cAMPaccumulation without significantly shifting the MCP-1 IC₅₀, a resultconsistent with the hypothesis that pertussis toxin treatmentfunctionally uncouples the MCP-1RB receptor from Gαi. These results alsosuggest that both the inhibition of adenylyl cyclase activity and themobilization of intracellular calcium may be mediated through activationof the same G-protein in the 293 cells.

D. Discussion of Results

MCP-1 induced a rapid rise in intracellular calcium in indo-1-loaded 293cells that were stably transfected with MCP-1RB. The stable cell linealso demonstrated dose-dependent homologous desensitization of calciummobilization in response to MCP-1. The relative contributions ofextracellular and intracellular calcium stores to this calcium flux hasbeen controversial. The results above support the conclusion that theinitial rise in cytoplasmic calcium after activation of the MCP-1receptor in 293 cells is almost exclusively due to the release ofintracellular calcium stores. First, chelation of extracellular calciumwith EGTA (2 mM to 10 mM) had little effect on the rise and peal levelsof the calcium transients, but did hasten the return to baseline calciumlevels. Second, the same result was obtained when the transfected cellswere incubated in calcium-free media, supplemented with 1 mM EGTA.Finally, virtually identical results were obtained in the presence of 5mM Ni²⁺, which blocks the influx of extracellular calcium.

Activation of the MCP-1RB receptor led to profound inhibition ofadenylyl cyclase, suggesting coupling via one of the isoforms of Gαi.Similar results were obtained using the cloned MIP-1α/RANTES receptor,indicating that at least two of the receptors for C-C chemokinesactivate Gαi. Moreover, pertussis toxin blocked both the calciummobilization as well as the inhibition of adenylyl cyclase induced byMCP-1. Similarity in the pertussis toxin dose-response curves forcalcium mobilization and inhibition of adenylyl cyclase suggests thatboth may be downstream consequences of coupling to Gαi. These studiesare the first demonstration of adenylyl cyclase inhibition by chemokinereceptors, and are consistent with reports that leukocyte chemotaxis toIL-8, fMLP and MCP-1 is sensitive to inhibition by pertussis toxin.Oppenheim, Ann. Rev. Immunol. 9: 617-48 (1991); Spangrude, J.Immunol.135: 4135-43 (1985); Sozzini, J. Immunol. 147: 2215-21 (1991).

Although inhibition of adenylyl cyclase is the most thoroughlycharacterized downstream effect of the activation of Gαi in leukocytes,Gαi has also been implicated in the activation of potassium channels, inthe induction of mitosis and in the activation of Ras and microtubuleassociated protein (MAP) kinase in fMLP stimulated neutrophils. Yatani,Nature 336:680-82 (1988); Seuwan, J. Biol. Chem. 265: 22292-99 (1990);Worthen, J. Clin. Invest. 94:815-23 (1994). Thus, activation of Gαi mayactivate a complex array of intracellular signals that ultimately leadto leukocyte activation and chemotaxis.

A pertussis toxin-sensitive signal transduction pathway in which βγdimers, released in conjunction with Gαi, activate the β₂ isoform of thephospholipase C (PLCβ₂) to generate IP₃ has been described. Wu, Science261:101-031. Cellular activation via this pathway would be expected toresult in a pertussis toxin-sensitive mobilization of intracellularcalcium. However, 293 cells stably expressing the recombinant MCP-1receptor hydrolyze little, if any PI (phosphatydlinositol) whenchallenged with MCP-1. In control experiments, Gq-coupled receptors,co-transfected into this cell line, increased total inositol phosphates5- to 9-fold upon activation. The failure to detect PI turnover in theMCP-1RB transfected cells suggests that the MCP-1 receptor mobilizesintracellular calcium via a novel mechanism independent of IP₃.

MCP-1RB was remarkably specific for MCP-1. In the cyclase assay the IC₅₀for inhibition by MCP-1 was 90 pM, whereas related chemokines wereineffective at up to 1 μM. In contrast, the MIP-1-α/RANTES receptor hasan IC50 of approximately 100 pM for MIP-1α and RANTES, and 10 nM and 820nM for MIP-1β and MCP-1, respectively. Thus, MCP-1 had a selectivity ofat least 9000-fold for the MCP-1 receptor, whereas MIP-1α and RANTES hada similar preference for the MIP-1-α/RANTES receptor, as compared toMCP-1RB. It is likely, therefore, that under physiological conditions,MCP-1, MIP-1α, and RANTES act as specific agonists of MCP-1RB and theMIP-1-α/RANTES receptor, respectively.

The IC₅₀ for MCP-1-medicated inhibition of adenylyl cyclase wasapproximately 90 pM, well below the dissociation constant for binding(K_(d) =260 pM) which suggests that relatively few receptors must beoccupied for efficient coupling to Gαi. In contrast, very high receptoroccupancy was required to elicit peak intracellular calcium fluxes (EC₅₀=2-4 nM). It is interesting to note, in this regard, that the EC₅₀ formonocyte chemotaxis to MCP-1 is subnanomolar. Yoshimura, J. Immunol.145:292-97 (1990). Thus the induction of chemotaxis, which is thehallmark function of MCP-1 is optimal at MCP-1 concentrations thatprovide for efficient coupling/signaling through Gαi but areinsufficient to elicit maximal intracellular calcium fluxes andsubsequent receptor desensitization, suggesting that modest increases inintracellular calcium are sufficient to initiate and support monocytechemotaxis. The high levels of intracellular calcium detected atnanomolar concentrations of MCP-1 may serve to stop monocyte migrationby desensitizing the receptor and unregulating adhesion molecules.

MCP-1 is synthesized and secreted in vitro by a number of differentcells in response to a variety of different cytokines or oxidativelymodified lipoproteins. The specificity of the cloned receptor for MCP-1,coupled with the fact that only monocytes, basophils, and a subset of Tlymphocytes response to MCP-1, provides for an effective means oflimiting the spectrum of infiltrating leukocytes in areas where MCP-1 isabundant. Early atherosclerotic lesions have a predominantly monocyticinfiltrate and MCP-1 is abundant in these lesions. In contrast, theMIP-1-α/RANTES receptor binds and signals in response to multiplechemokines, and may serve to mediate more complex inflammatoryreactions. Once activated, however, the MCP-1 and MIP-1-α/RANTESreceptors appear to use similar signal transduction pathways.

Dose response curves generated in the calcium fluorimetric and adenylylcyclase inhibition assays were fit by a nonlinear least squares programto the logistic equation:

    Effect=max effect/[1+(EC.sub.50 /(agonist).sup.n)]

where n and EC₅₀ represent the Hill coefficient and the agonistconcentration that elicited a half-maximal response, respectively, andwere derived from the fitted curve. Curve fitting was done with thecomputer program "Prism" (by Graph Pad, San Diego, Calif.). Resultsrepresent the mean ±SE. The 95% confidence intervals (CI) of the EC₅₀and IC₅₀ values, when given, were calculated from the log EC₅₀ and IC₅₀values, respectively.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 14                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2232 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 40..1161                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GGATTGAACA AGGACGCATT TCCCCAGTAC ATCCACAAC ATG CTG TCC - # ACA TCT            54                                                                                        - #                  - #       Met Leu Ser Thr Ser                            - #                  - #         1         - #      5        - - CGT TCT CGG TTT ATC AGA AAT ACC AAC GAG AG - #C GGT GAA GAA GTC ACC          102                                                                       Arg Ser Arg Phe Ile Arg Asn Thr Asn Glu Se - #r Gly Glu Glu Val Thr                            10 - #                 15 - #                 20              - - ACC TTT TTT GAT TAT GAT TAC GGT GCT CCC TG - #T CAT AAA TTT GAC GTG          150                                                                       Thr Phe Phe Asp Tyr Asp Tyr Gly Ala Pro Cy - #s His Lys Phe Asp Val                        25     - #             30     - #             35                  - - AAG CAA ATT GGG GCC CAA CTC CTG CCT CCG CT - #C TAC TCG CTG GTG TTC          198                                                                       Lys Gln Ile Gly Ala Gln Leu Leu Pro Pro Le - #u Tyr Ser Leu Val Phe                    40         - #         45         - #         50                      - - ATC TTT GGT TTT GTG GGC AAC ATG CTG GTC GT - #C CTC ATC TTA ATA AAC          246                                                                       Ile Phe Gly Phe Val Gly Asn Met Leu Val Va - #l Leu Ile Leu Ile Asn                55             - #     60             - #     65                          - - TGC AAA AAG CTG AAG TGC TTG ACT GAC ATT TA - #C CTG CTC AAC CTG GCC          294                                                                       Cys Lys Lys Leu Lys Cys Leu Thr Asp Ile Ty - #r Leu Leu Asn Leu Ala            70                 - # 75                 - # 80                 - # 85       - - ATC TCT GAT CTG CTT TTT CTT ATT ACT CTC CC - #A TTG TGG GCT CAC TCT          342                                                                       Ile Ser Asp Leu Leu Phe Leu Ile Thr Leu Pr - #o Leu Trp Ala His Ser                            90 - #                 95 - #                100              - - GCT GCA AAT GAG TGG GTC TTT GGG AAT GCA AT - #G TGC AAA TTA TTC ACA          390                                                                       Ala Ala Asn Glu Trp Val Phe Gly Asn Ala Me - #t Cys Lys Leu Phe Thr                       105      - #           110      - #           115                  - - GGG CTG TAT CAC ATC GGT TAT TTT GGC GGA AT - #C TTC TTC ATC ATC CTC          438                                                                       Gly Leu Tyr His Ile Gly Tyr Phe Gly Gly Il - #e Phe Phe Ile Ile Leu                   120          - #       125          - #       130                      - - CTG ACA ATC GAT AGA TAC CTG GCT ATT GTC CA - #T GCT GTG TTT GCT TTA          486                                                                       Leu Thr Ile Asp Arg Tyr Leu Ala Ile Val Hi - #s Ala Val Phe Ala Leu               135              - #   140              - #   145                          - - AAA GCC AGG ACG GTC ACC TTT GGG GTG GTG AC - #A AGT GTG ATC ACC TGG          534                                                                       Lys Ala Arg Thr Val Thr Phe Gly Val Val Th - #r Ser Val Ile Thr Trp           150                 1 - #55                 1 - #60                 1 -      #65                                                                              - - TTG GTG GCT GTG TTT GCT TCT GTC CCA GGA AT - #C ATC TTT ACT AAA        TGC      582                                                                    Leu Val Ala Val Phe Ala Ser Val Pro Gly Il - #e Ile Phe Thr Lys Cys                          170  - #               175  - #               180              - - CAG AAA GAA GAT TCT GTT TAT GTC TGT GGC CC - #T TAT TTT CCA CGA GGA          630                                                                       Gln Lys Glu Asp Ser Val Tyr Val Cys Gly Pr - #o Tyr Phe Pro Arg Gly                       185      - #           190      - #           195                  - - TGG AAT AAT TTC CAC ACA ATA ATG AGG AAC AT - #T TTG GGG CTG GTC CTG          678                                                                       Trp Asn Asn Phe His Thr Ile Met Arg Asn Il - #e Leu Gly Leu Val Leu                   200          - #       205          - #       210                      - - CCG CTG CTC ATC ATG GTC ATC TGC TAC TCG GG - #A ATC CTG AAA ACC CTG          726                                                                       Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gl - #y Ile Leu Lys Thr Leu               215              - #   220              - #   225                          - - CTT CGG TGT CGA AAC GAG AAG AAG AGG CAT AG - #G GCA GTG AGA GTC ATC          774                                                                       Leu Arg Cys Arg Asn Glu Lys Lys Arg His Ar - #g Ala Val Arg Val Ile           230                 2 - #35                 2 - #40                 2 -      #45                                                                              - - TTC ACC ATC ATG ATT GTT TAC TTT CTC TTC TG - #G ACT CCC TAT AAC        ATT      822                                                                    Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Tr - #p Thr Pro Tyr Asn Ile                          250  - #               255  - #               260              - - GTC ATT CTC CTG AAC ACC TTC CAG GAA TTC TT - #C GGC CTG AGT AAC TGT          870                                                                       Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Ph - #e Gly Leu Ser Asn Cys                       265      - #           270      - #           275                  - - GAA AGC ACC AGT CAA CTG GAC CAA GCC ACG CA - #G GTG ACA GAG ACT CTT          918                                                                       Glu Ser Thr Ser Gln Leu Asp Gln Ala Thr Gl - #n Val Thr Glu Thr Leu                   280          - #       285          - #       290                      - - GGG ATG ACT CAC TGC TGC ATC AAT CCC ATC AT - #C TAT GCC TTC GTT GGG          966                                                                       Gly Met Thr His Cys Cys Ile Asn Pro Ile Il - #e Tyr Ala Phe Val Gly               295              - #   300              - #   305                          - - GAG AAG TTC AGA AGC CTT TTT CAC ATA GCT CT - #T GGC TGT AGG ATT GCC         1014                                                                       Glu Lys Phe Arg Ser Leu Phe His Ile Ala Le - #u Gly Cys Arg Ile Ala           310                 3 - #15                 3 - #20                 3 -      #25                                                                              - - CCA CTC CAA AAA CCA GTG TGT GGA GGT CCA GG - #A GTG AGA CCA GGA        AAG     1062                                                                    Pro Leu Gln Lys Pro Val Cys Gly Gly Pro Gl - #y Val Arg Pro Gly Lys                          330  - #               335  - #               340              - - AAT GTG AAA GTG ACT ACA CAA GGA CTC CTC GA - #T GGT CGT GGA AAA GGA         1110                                                                       Asn Val Lys Val Thr Thr Gln Gly Leu Leu As - #p Gly Arg Gly Lys Gly                       345      - #           350      - #           355                  - - AAG TCA ATT GGC AGA GCC CCT GAA GCC AGT CT - #T CAG GAC AAA GAA GGA         1158                                                                       Lys Ser Ile Gly Arg Ala Pro Glu Ala Ser Le - #u Gln Asp Lys Glu Gly                   360          - #       365          - #       370                      - - GCC TAGAGACAGA AATGACAGAT CTCTGCTTTG GAAATCACAC GTCTGGCTT - #C              1211                                                                       Ala                                                                            - - ACAGATGTGT GATTCACAGT GTGAATCTTG GTGTCTACGT TACCAGGCAG GA -             #AGGCTGAG   1271                                                                 - - AGGAGAGAGA CTCCAGCTGG GTTGGAAAAC AGTATTTTCC AAACTACCTT CC -            #AGTTCCTC   1331                                                                 - - ATTTTTGAAT ACAGGCATAG AGTTCAGACT TTTTTTAAAT AGTAAAAATA AA -            #ATTAAAGC   1391                                                                 - - TGAAAACTGC AACTTGTAAA TGTGGTAAAG AGTTAGTTTG AGTTGCTATC AT -            #GTCAAACG   1451                                                                 - - TGAAAATGCT GTATTAGTCA CAGAGATAAT TCTAGCTTTG AGCTTAAGAA TT -            #TTGAGCAG   1511                                                                 - - GTGGTATGTT TGGGAGACTG CTGAGTCAAC CCAATAGTTG TTGATTGGCA GG -            #AGTTGGAA   1571                                                                 - - GTGTGTGATC TGTGGGCACA TTAGCCTATG TGCATGCAGC ATCTAAGTAA TG -            #ATGTCGTT   1631                                                                 - - TGAATCACAG TATACGCTCC ATCGCTGTCA TCTCAGCTGG ATCTCCATTC TC -            #TCAGGCTT   1691                                                                 - - GCTGCCAAAA GCCTTTTGTG TTTTGTTTTG TATCATTATG AAGTCATGCG TT -            #TAATCACA   1751                                                                 - - TTCGAGTGTT TCAGTGCTTC GCAGATGTCC TTGATGCTCA TATTGTTCCC TA -            #ATTTGCCA   1811                                                                 - - GTGGGAACTC CTAAATCAAA TTGGCTTCTA ATCAAAGCTT TTAAACCCTA TT -            #GGTAAAGA   1871                                                                 - - ATGGAAGGTG GAGAAGCTCC CTGAAGTAAG CAAAGACTTT CCTCTTAGTC GA -            #GCCAAGTT   1931                                                                 - - AAGAATGTTC TTATGTTGCC CAGTGTGTTT CTGATCTGAT GCAAGCAAGA AA -            #CACTGGGC   1991                                                                 - - TTCTAGAACC AGGCAACTTG GGAACTAGAC TCCCAAGCTG GACTATGGCT CT -            #ACTTTCAG   2051                                                                 - - GCCACATGGC TAAAGAAGGT TTCAGAAAGA AGTGGGGACA GAGCAGAACT TT -            #CACCTTCA   2111                                                                 - - TATATTTGTA TGATCCTAAT GAATGCATAA AATGTTAAGT TGATGGTGAT GA -            #AATGTAAA   2171                                                                 - - TACTGTTTTT AACAACTATG ATTTGGAAAA TAAATCAATG CTATAACTAT GT -            #TGATAAAA   2231                                                                 - - G                  - #                  - #                  - #                 2232                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 374 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Ar - #g Asn Thr Asn Glu Ser        1               5 - #                 10 - #                 15              - - Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr As - #p Tyr Gly Ala Pro Cys                   20     - #             25     - #             30                  - - His Lys Phe Asp Val Lys Gln Ile Gly Ala Gl - #n Leu Leu Pro Pro Leu               35         - #         40         - #         45                      - - Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gl - #y Asn Met Leu Val Val           50             - #     55             - #     60                          - - Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cy - #s Leu Thr Asp Ile Tyr       65                 - # 70                 - # 75                 - # 80       - - Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Ph - #e Leu Ile Thr Leu Pro                       85 - #                 90 - #                 95              - - Leu Trp Ala His Ser Ala Ala Asn Glu Trp Va - #l Phe Gly Asn Ala Met                  100      - #           105      - #           110                  - - Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gl - #y Tyr Phe Gly Gly Ile              115          - #       120          - #       125                      - - Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Ty - #r Leu Ala Ile Val His          130              - #   135              - #   140                          - - Ala Val Phe Ala Leu Lys Ala Arg Thr Val Th - #r Phe Gly Val Val Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Val Ile Thr Trp Leu Val Ala Val Phe Al - #a Ser Val Pro Gly        Ile                                                                                             165  - #               170  - #               175             - - Ile Phe Thr Lys Cys Gln Lys Glu Asp Ser Va - #l Tyr Val Cys Gly Pro                  180      - #           185      - #           190                  - - Tyr Phe Pro Arg Gly Trp Asn Asn Phe His Th - #r Ile Met Arg Asn Ile              195          - #       200          - #       205                      - - Leu Gly Leu Val Leu Pro Leu Leu Ile Met Va - #l Ile Cys Tyr Ser Gly          210              - #   215              - #   220                          - - Ile Leu Lys Thr Leu Leu Arg Cys Arg Asn Gl - #u Lys Lys Arg His Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Val Arg Val Ile Phe Thr Ile Met Ile Va - #l Tyr Phe Leu Phe        Trp                                                                                             245  - #               250  - #               255             - - Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Th - #r Phe Gln Glu Phe Phe                  260      - #           265      - #           270                  - - Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Le - #u Asp Gln Ala Thr Gln              275          - #       280          - #       285                      - - Val Thr Glu Thr Leu Gly Met Thr His Cys Cy - #s Ile Asn Pro Ile Ile          290              - #   295              - #   300                          - - Tyr Ala Phe Val Gly Glu Lys Phe Arg Ser Le - #u Phe His Ile Ala Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Cys Arg Ile Ala Pro Leu Gln Lys Pro Va - #l Cys Gly Gly Pro        Gly                                                                                             325  - #               330  - #               335             - - Val Arg Pro Gly Lys Asn Val Lys Val Thr Th - #r Gln Gly Leu Leu Asp                  340      - #           345      - #           350                  - - Gly Arg Gly Lys Gly Lys Ser Ile Gly Arg Al - #a Pro Glu Ala Ser Leu              355          - #       360          - #       365                      - - Gln Asp Lys Glu Gly Ala                                                      370                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1979 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 81..1160                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CAGGACTGCC TGAGACAAGC CACAAGCTGA ACAGAGAAAG TGGATTGAAC AA -             #GGACGCAT     60                                                                 - - TTCCCCAGTA CATCCACAAC ATG CTG TCC ACA TCT CGT TC - #T CGG TTT ATC            110                                                                                        - #    Met Leu Ser Thr Ser Arg Ser Arg - #Phe Ile                             - #      1            - #   5               - #   10         - - AGA AAT ACC AAC GAG AGC GGT GAA GAA GTC AC - #C ACC TTT TTT GAT TAT          158                                                                       Arg Asn Thr Asn Glu Ser Gly Glu Glu Val Th - #r Thr Phe Phe Asp Tyr                            15 - #                 20 - #                 25              - - GAT TAC GGT GCT CCC TGT CAT AAA TTT GAC GT - #G AAG CAA ATT GGG GCC          206                                                                       Asp Tyr Gly Ala Pro Cys His Lys Phe Asp Va - #l Lys Gln Ile Gly Ala                        30     - #             35     - #             40                  - - CAA CTC CTG CCT CCG CTC TAC TCG CTG GTG TT - #C ATC TTT GGT TTT GTG          254                                                                       Gln Leu Leu Pro Pro Leu Tyr Ser Leu Val Ph - #e Ile Phe Gly Phe Val                    45         - #         50         - #         55                      - - GGC AAC ATG CTG GTC GTC CTC ATC TTA ATA AA - #C TGC AAA AAG CTG AAG          302                                                                       Gly Asn Met Leu Val Val Leu Ile Leu Ile As - #n Cys Lys Lys Leu Lys                60             - #     65             - #     70                          - - TGC TTG ACT GAC ATT TAC CTG CTC AAC CTG GC - #C ATC TCT GAT CTG CTT          350                                                                       Cys Leu Thr Asp Ile Tyr Leu Leu Asn Leu Al - #a Ile Ser Asp Leu Leu            75                 - # 80                 - # 85                 - # 90       - - TTT CTT ATT ACT CTC CCA TTG TGG GCT CAC TC - #T GCT GCA AAT GAG TGG          398                                                                       Phe Leu Ile Thr Leu Pro Leu Trp Ala His Se - #r Ala Ala Asn Glu Trp                            95 - #                100 - #                105              - - GTC TTT GGG AAT GCA ATG TGC AAA TTA TTC AC - #A GGG CTG TAT CAC ATC          446                                                                       Val Phe Gly Asn Ala Met Cys Lys Leu Phe Th - #r Gly Leu Tyr His Ile                       110      - #           115      - #           120                  - - GGT TAT TTT GGC GGA ATC TTC TTC ATC ATC CT - #C CTG ACA ATC GAT AGA          494                                                                       Gly Tyr Phe Gly Gly Ile Phe Phe Ile Ile Le - #u Leu Thr Ile Asp Arg                   125          - #       130          - #       135                      - - TAC CTG GCT ATT GTC CAT GCT GTG TTT GCT TT - #A AAA GCC AGG ACG GTC          542                                                                       Tyr Leu Ala Ile Val His Ala Val Phe Ala Le - #u Lys Ala Arg Thr Val               140              - #   145              - #   150                          - - ACC TTT GGG GTG GTG ACA AGT GTG ATC ACC TG - #G TTG GTG GCT GTG TTT          590                                                                       Thr Phe Gly Val Val Thr Ser Val Ile Thr Tr - #p Leu Val Ala Val Phe           155                 1 - #60                 1 - #65                 1 -      #70                                                                              - - GCT TCT GTC CCA GGA ATC ATC TTT ACT AAA TG - #C CAG AAA GAA GAT        TCT      638                                                                    Ala Ser Val Pro Gly Ile Ile Phe Thr Lys Cy - #s Gln Lys Glu Asp Ser                          175  - #               180  - #               185              - - GTT TAT GTC TGT GGC CCT TAT TTT CCA CGA GG - #A TGG AAT AAT TTC CAC          686                                                                       Val Tyr Val Cys Gly Pro Tyr Phe Pro Arg Gl - #y Trp Asn Asn Phe His                       190      - #           195      - #           200                  - - ACA ATA ATG AGG AAC ATT TTG GGG CTG GTC CT - #G CCG CTG CTC ATC ATG          734                                                                       Thr Ile Met Arg Asn Ile Leu Gly Leu Val Le - #u Pro Leu Leu Ile Met                   205          - #       210          - #       215                      - - GTC ATC TGC TAC TCG GGA ATC CTG AAA ACC CT - #G CTT CGG TGT CGA AAC          782                                                                       Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Le - #u Leu Arg Cys Arg Asn               220              - #   225              - #   230                          - - GAG AAG AAG AGG CAT AGG GCA GTG AGA GTC AT - #C TTC ACC ATC ATG ATT          830                                                                       Glu Lys Lys Arg His Arg Ala Val Arg Val Il - #e Phe Thr Ile Met Ile           235                 2 - #40                 2 - #45                 2 -      #50                                                                              - - GTT TAC TTT CTC TTC TGG ACT CCC TAT AAC AT - #T GTC ATT CTC CTG        AAC      878                                                                    Val Tyr Phe Leu Phe Trp Thr Pro Tyr Asn Il - #e Val Ile Leu Leu Asn                          255  - #               260  - #               265              - - ACC TTC CAG GAA TTC TTC GGC CTG AGT AAC TG - #T GAA AGC ACC AGT CAA          926                                                                       Thr Phe Gln Glu Phe Phe Gly Leu Ser Asn Cy - #s Glu Ser Thr Ser Gln                       270      - #           275      - #           280                  - - CTG GAC CAA GCC ACG CAG GTG ACA GAG ACT CT - #T GGG ATG ACT CAC TGC          974                                                                       Leu Asp Gln Ala Thr Gln Val Thr Glu Thr Le - #u Gly Met Thr His Cys                   285          - #       290          - #       295                      - - TGC ATC AAT CCC ATC ATC TAT GCC TTC GTT GG - #G GAG AAG TTC AGA AGG         1022                                                                       Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gl - #y Glu Lys Phe Arg Arg               300              - #   305              - #   310                          - - TAT CTC TCG GTG TTC TTC CGA AAG CAC ATC AC - #C AAG CGC TTC TGC AAA         1070                                                                       Tyr Leu Ser Val Phe Phe Arg Lys His Ile Th - #r Lys Arg Phe Cys Lys           315                 3 - #20                 3 - #25                 3 -      #30                                                                              - - CAA TGT CCA GTT TTC TAC AGG GAG ACA GTG GA - #T GGA GTG ACT TCA        ACA     1118                                                                    Gln Cys Pro Val Phe Tyr Arg Glu Thr Val As - #p Gly Val Thr Ser Thr                          335  - #               340  - #               345              - - AAC ACG CCT TCC ACT GGG GAG CAG GAA GTC TC - #G GCT GGT TTA                 - #1160                                                                    Asn Thr Pro Ser Thr Gly Glu Gln Glu Val Se - #r Ala Gly Leu                               350      - #           355      - #           360                  - - TAAAACGAGG AGCAGTTTGA TTGTTGTTTA TAAAGGGAGA TAACAATCTG TA -             #TATAACAA   1220                                                                 - - CAAACTTCAA GGGTTTGTTG AACAATAGAA ACCTGTAAAG CAGGTGCCCA GG -            #AACCTCAG   1280                                                                 - - GGCTGTGTGT ACTAATACAG ACTATGTCAC CCAATGCATA TCCAACATGT GC -            #TCAGGGAA   1340                                                                 - - TAATCCAGAA AAACTGTGGG TAGAGACTTT GACTCTCCAG AAAGCTCATC TC -            #AGCTCCTG   1400                                                                 - - AAAAATGCCT CATTACCTTG TGCTAATCCT CTTTTTCTAG TCTTCATAAT TT -            #CTTCACTC   1460                                                                 - - AATCTCTGAT TCTGTCAATG TCTTGAAATC AAGGGCCAGC TGGAGGTGAA GA -            #AGAGAATG   1520                                                                 - - TGACAGGCAC AGATGAATGG GAGTGAGGGA TAGTGGGGTC AGGGCTGAGA GG -            #AGAAGGAG   1580                                                                 - - GGAGACATGA GCATGGCTGA GCCTGGACAA AGACAAAGGT GAGCAAAGGG CT -            #CACGCATT   1640                                                                 - - CAGCCAGGAG ATGATACTGG TCCTTAGCCC CATCTGCCAC GTGTATTTAA CC -            #TTGAAGGG   1700                                                                 - - TTCACCAGGT CAGGGAGAGT TTGGGAACTG CAATAACCTG GGAGTTTTGG TG -            #GAGTCCGA   1760                                                                 - - TGATTCTCTT TTGCATAAGT GCATGACATA TTTTTGCTTT ATTACAGTTT AT -            #CTATGGCA   1820                                                                 - - CCCATGCACC TTACATTTGA AATCTATGAA ATATCATGCT CCATTGTTCA GA -            #TGCTTCTT   1880                                                                 - - AGGCCACATC CCCCTGTCTA AAAATTCAGA AAATTTTTGT TTATAAAAGA TG -            #CATTATCT   1940                                                                 - - ATGATATGCT AATATATGTA TATGCAATAT AAAATTTAG      - #                      - #  1979                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Ar - #g Asn Thr Asn Glu Ser        1               5 - #                 10 - #                 15              - - Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr As - #p Tyr Gly Ala Pro Cys                   20     - #             25     - #             30                  - - His Lys Phe Asp Val Lys Gln Ile Gly Ala Gl - #n Leu Leu Pro Pro Leu               35         - #         40         - #         45                      - - Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gl - #y Asn Met Leu Val Val           50             - #     55             - #     60                          - - Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cy - #s Leu Thr Asp Ile Tyr       65                 - # 70                 - # 75                 - # 80       - - Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Ph - #e Leu Ile Thr Leu Pro                       85 - #                 90 - #                 95              - - Leu Trp Ala His Ser Ala Ala Asn Glu Trp Va - #l Phe Gly Asn Ala Met                  100      - #           105      - #           110                  - - Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gl - #y Tyr Phe Gly Gly Ile              115          - #       120          - #       125                      - - Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Ty - #r Leu Ala Ile Val His          130              - #   135              - #   140                          - - Ala Val Phe Ala Leu Lys Ala Arg Thr Val Th - #r Phe Gly Val Val Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Val Ile Thr Trp Leu Val Ala Val Phe Al - #a Ser Val Pro Gly        Ile                                                                                             165  - #               170  - #               175             - - Ile Phe Thr Lys Cys Gln Lys Glu Asp Ser Va - #l Tyr Val Cys Gly Pro                  180      - #           185      - #           190                  - - Tyr Phe Pro Arg Gly Trp Asn Asn Phe His Th - #r Ile Met Arg Asn Ile              195          - #       200          - #       205                      - - Leu Gly Leu Val Leu Pro Leu Leu Ile Met Va - #l Ile Cys Tyr Ser Gly          210              - #   215              - #   220                          - - Ile Leu Lys Thr Leu Leu Arg Cys Arg Asn Gl - #u Lys Lys Arg His Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Val Arg Val Ile Phe Thr Ile Met Ile Va - #l Tyr Phe Leu Phe        Trp                                                                                             245  - #               250  - #               255             - - Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Th - #r Phe Gln Glu Phe Phe                  260      - #           265      - #           270                  - - Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Le - #u Asp Gln Ala Thr Gln              275          - #       280          - #       285                      - - Val Thr Glu Thr Leu Gly Met Thr His Cys Cy - #s Ile Asn Pro Ile Ile          290              - #   295              - #   300                          - - Tyr Ala Phe Val Gly Glu Lys Phe Arg Arg Ty - #r Leu Ser Val Phe Phe      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Arg Lys His Ile Thr Lys Arg Phe Cys Lys Gl - #n Cys Pro Val Phe        Tyr                                                                                             325  - #               330  - #               335             - - Arg Glu Thr Val Asp Gly Val Thr Ser Thr As - #n Thr Pro Ser Thr Gly                  340      - #           345      - #           350                  - - Glu Gln Glu Val Ser Ala Gly Leu                                                  355          - #       360                                             - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 355 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Glu Thr Pro Asn Thr Thr Glu Asp Tyr As - #p Thr Thr Thr Glu Phe        1               5 - #                 10 - #                 15              - - Asp Tyr Gly Asp Ala Thr Pro Cys Gln Lys Va - #l Asn Glu Arg Ala Phe                  20      - #            25      - #            30                   - - Gly Ala Gln Leu Leu Pro Pro Leu Tyr Ser Le - #u Val Phe Val Ile Gly               35         - #         40         - #         45                      - - Leu Val Gly Asn Ile Leu Val Val Leu Val Le - #u Val Gln Tyr Lys Arg           50             - #     55             - #     60                          - - Leu Lys Asn Met Thr Ser Ile Tyr Leu Leu As - #n Leu Ala Ile Ser Asp       65                 - # 70                 - # 75                 - # 80       - - Leu Leu Phe Leu Phe Thr Leu Pro Phe Trp Il - #e Asp Tyr Lys Leu Lys                       85 - #                 90 - #                 95              - - Asp Asp Trp Val Phe Gly Asp Ala Met Cys Ly - #s Ile Leu Ser Gly Phe                  100      - #           105      - #           110                  - - Tyr Tyr Thr Gly Leu Tyr Ser Glu Ile Phe Ph - #e Ile Ile Leu Leu Thr              115          - #       120          - #       125                      - - Ile Asp Arg Tyr Leu Ala Ile Val His Ala Va - #l Phe Ala Leu Arg Ala          130              - #   135              - #   140                          - - Arg Thr Val Thr Phe Gly Val Ile Thr Ser Il - #e Ile Ile Trp Ala Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Ile Leu Ala Ser Met Pro Gly Leu Tyr Ph - #e Ser Lys Thr Gln        Trp                                                                                             165  - #               170  - #               175             - - Glu Phe Thr His His Thr Cys Ser Leu His Ph - #e Pro His Glu Ser Leu                  180      - #           185      - #           190                  - - Arg Glu Trp Lys Leu Phe Gln Ala Leu Lys Le - #u Asn Leu Phe Gly Leu              195          - #       200          - #       205                      - - Val Leu Pro Leu Leu Val Met Ile Ile Cys Ty - #r Thr Gly Ile Ile Lys          210              - #   215              - #   220                          - - Ile Leu Leu Arg Arg Pro Asn Glu Lys Lys Se - #r Lys Ala Val Arg Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Phe Val Ile Met Ile Ile Phe Phe Leu Ph - #e Trp Thr Pro Tyr        Asn                                                                                             245  - #               250  - #               255             - - Leu Thr Ile Leu Ile Ser Val Phe Gln Asp Ph - #e Leu Phe Thr His Glu                  260      - #           265      - #           270                  - - Cys Glu Gln Ser Arg His Leu Asp Leu Ala Va - #l Gln Val Thr Glu Val              275          - #       280          - #       285                      - - Ile Ala Tyr Thr His Cys Cys Val Asn Pro Va - #l Ile Tyr Ala Phe Val          290              - #   295              - #   300                          - - Gly Glu Arg Phe Arg Lys Tyr Leu Arg Gln Le - #u Phe His Arg Arg Val      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Val His Leu Val Lys Trp Leu Pro Phe Le - #u Ser Val Asp Arg        Leu                                                                                             325  - #               330  - #               335             - - Glu Arg Val  Ser Ser Thr Ser Pro Ser Thr - #Gly Glu His Glu Leu Ser                   340     - #            345     - #            350                 - - Ala Gly Phe                                                                      355                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 352 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Glu Gly Ile Ser Ile Tyr Thr Ser Asp As - #n Tyr Thr Glu Glu Met      1               5   - #                10  - #                15               - - Gly Ser Gly Asp Tyr Asp Ser Met Lys Glu Pr - #o Cys Phe Arg Glu Glu                  20      - #            25      - #            30                   - - Asn Ala Asn Phe Asn Lys Ile Phe Leu Pro Ty - #r Ile Tyr Ser Ile Ile              35          - #        40          - #        45                       - - Phe Leu Tyr Gly Ile Val Gly Asn Gly Leu Va - #l Ile Leu Val Met Gly          50              - #    55              - #    60                           - - Tyr Gln Lys Lys Leu Arg Ser Met Thr Asp Ly - #s Tyr Arg Leu His Leu      65                  - #70                  - #75                  - #80        - - Ser Val Ala Asp Leu Leu Phe Val Ile Thr Le - #u Pro Phe Trp Ala Val                      85  - #                90  - #                95               - - Asp Ala Val Ala Asn Trp Tyr Phe Gly Asn Ph - #e Leu Cys Lys Ala Val                  100      - #           105      - #           110                  - - His Val Ile Tyr Thr Val Asn Leu Tyr Ser Se - #r Val Leu Ile Leu Ala              115          - #       120          - #       125                      - - Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Va - #l His Ala Thr Asn Ser          130              - #   135              - #   140                          - - Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Va - #l Val Tyr Val Gly Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro As - #p Phe Ile Phe Ala        Asn                                                                                             165  - #               170  - #               175             - - Val Ser Glu Ala Asp Asp Arg Tyr Ile Cys As - #p Arg Phe Tyr Pro Asn                  180      - #           185      - #           190                  - - Asp Leu Trp Val Val Val Phe Gln Phe Gln Hi - #s Ile Met Val Gly Leu              195          - #       200          - #       205                      - - Ile Leu Pro Gly Ile Val Ile Leu Phe Cys Ty - #r Cys Ile Ile Ile Ser          210              - #   215              - #   220                          - - Lys Leu Ser His Ser Lys Gly His Gln Lys Ar - #g Lys Ala Leu Lys Tyr      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Tyr Val Ile Leu Ile Leu Ala Phe Phe Ala Cy - #s Trp Leu Pro Tyr        Tyr                                                                                             245  - #               250  - #               255             - - Ile Gly Ile Ser Ile Asp Ser Phe Ile Leu Le - #u Glu Ile Ile Lys Gln                  260      - #           265      - #           270                  - - Gly Cys Glu Phe Glu Asn Thr Val His Lys Tr - #p Ile Ser Ile Thr Glu              275          - #       280          - #       285                      - - Ala Leu Ala Phe Phe His Cys Cys Leu Asn Pr - #o Ile Leu Tyr Ala Phe          290              - #   295              - #   300                          - - Leu Gly Ala Lys Phe Lys Tyr Ser Ala Gln Hi - #s Ala Leu Thr Ser Val      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser Arg Gly Ser Ser Leu Lys Ile Leu Ser Ly - #s Gly Lys Arg Gly        Gly                                                                                             325  - #               330  - #               335             - - His Ser Ser Val Ser Thr Glu Ser Glu Ser Se - #r Ser Phe His Ser Ser                  340      - #           345      - #           350                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 350 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Met Ser Asn Ile Thr Asp Pro Gln Met Trp As - #p Phe Asp Asp Leu Asn      1               5   - #                10  - #                15               - - Phe Thr Gly Met Pro Pro Ala Asp Glu Asp Ty - #r Ser Pro Cys Met Leu                  20      - #            25      - #            30                   - - Glu Thr Glu Thr Leu Asn Lys Tyr Val Val Il - #e Ile Ala Tyr Ala Leu              35          - #        40          - #        45                       - - Val Phe Leu Leu Ser Leu Leu Gly Asn Ser Le - #u Val Met Leu Val Ile          50              - #    55              - #    60                           - - Leu Tyr Ser Arg Val Gly Arg Ser Val Thr As - #p Val Tyr Leu Leu Asn      65                  - #70                  - #75                  - #80        - - Leu Ala Leu Ala Asp Leu Leu Phe Ala Leu Th - #r Leu Pro Ile Trp Ala                      85  - #                90  - #                95               - - Ala Ser Lys Val Asn Gly Trp Ile Phe Gly Th - #r Phe Leu Cys Lys Val                  100      - #           105      - #           110                  - - Val Ser Leu Leu Lys Glu Val Asn Phe Tyr Se - #r Gly Ile Leu Leu Leu              115          - #       120          - #       125                      - - Ala Cys Ile Ser Val Asp Arg Tyr Leu Ala Il - #e Val His Ala Thr Arg          130              - #   135              - #   140                          - - Thr Leu Thr Gln Lys Arg His Leu Val Lys Ph - #e Val Cys Leu Gly Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Trp Gly Leu Ser Met Asn Leu Ser Leu Pro Ph - #e Phe Leu Phe Arg        Gln                                                                                             165  - #               170  - #               175             - - Ala Tyr His Pro Asn Asn Ser Ser Pro Val Cy - #s Tyr Glu Val Leu Gly                  180      - #           185      - #           190                  - - Asn Asp Thr Ala Lys Trp Arg Met Val Leu Ar - #g Ile Leu Pro His Thr              195          - #       200          - #       205                      - - Phe Gly Phe Ile Val Pro Leu Phe Val Met Le - #u Phe Cys Tyr Gly Phe          210              - #   215              - #   220                          - - Thr Leu Arg Thr Leu Phe Lys Ala His Met Gl - #y Gln Lys His Arg Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Met Arg Val Ile Phe Ala Val Val Leu Ile Ph - #e Leu Leu Cys Trp        Leu                                                                                             245  - #               250  - #               255             - - Pro Tyr Asn Leu Val Leu Leu Ala Asp Thr Le - #u Met Arg Thr Gln Val                  260      - #           265      - #           270                  - - Ile Gln Glu Thr Cys Glu Arg Arg Asn Asn Il - #e Gly Arg Ala Leu Asp              275          - #       280          - #       285                      - - Ala Thr Glu Ile Leu Gly Phe Leu His Ser Cy - #s Leu Asn Pro Ile Ile          290              - #   295              - #   300                          - - Tyr Ala Phe Ile Gly Gln Asn Phe Arg His Gl - #y Phe Leu Lys Ile Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Met His Gly Leu Val Ser Lys Glu Phe Le - #u Ala Arg His Arg        Val                                                                                             325  - #               330  - #               335             - - Thr Ser Tyr Thr Ser Ser Ser Val Asn Val Se - #r Ser Asn Leu                          340      - #           345      - #           350                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 355 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Glu Ser Asp Ser Phe Glu Asp Phe Trp Ly - #s Gly Glu Asp Leu Ser      1               5   - #                10  - #                15               - - Asn Tyr Ser Tyr Ser Ser Thr Leu Pro Pro Ph - #e Leu Leu Asp Ala Ala                  20      - #            25      - #            30                   - - Pro Cys Glu Pro Glu Ser Leu Glu Ile Asn Ly - #s Tyr Phe Val Val Ile              35          - #        40          - #        45                       - - Ile Tyr Ala Leu Val Phe Leu Leu Ser Leu Le - #u Gly Asn Ser Leu Val          50              - #    55              - #    60                           - - Met Leu Val Ile Leu Tyr Ser Arg Val Gly Ar - #g Ser Val Thr Asp Val      65                  - #70                  - #75                  - #80        - - Tyr Leu Leu Asn Leu Ala Leu Ala Asp Leu Le - #u Phe Ala Leu Thr Leu                      85  - #                90  - #                95               - - Pro Ile Trp Ala Ala Ser Lys Val Asn Gly Tr - #p Ile Phe Gly Thr Phe                  100      - #           105      - #           110                  - - Leu Cys Lys Val Val Ser Leu Leu Lys Glu Va - #l Asn Phe Tyr Ser Gly              115          - #       120          - #       125                      - - Ile Leu Leu Leu Ala Cys Ile Ser Val Asp Ar - #g Tyr Leu Ala Ile Val          130              - #   135              - #   140                          - - His Ala Thr Arg Thr Leu Thr Gln Lys Arg Ty - #r Leu Val Lys Phe Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Leu Ser Ile Trp Gly Leu Ser Leu Leu Le - #u Ala Leu Pro Val        Leu                                                                                             165  - #               170  - #               175             - - Leu Phe Arg Arg Thr Val Tyr Ser Ser Asn Va - #l Ser Pro Ala Cys Tyr                  180      - #           185      - #           190                  - - Glu Asp Met Gly Asn Asn Thr Ala Asn Trp Ar - #g Met Leu Leu Arg Ile              195          - #       200          - #       205                      - - Leu Pro Gln Ser Phe Gly Phe Ile Val Pro Le - #u Leu Ile Met Leu Phe          210              - #   215              - #   220                          - - Cys Tyr Gly Phe Thr Leu Arg Thr Leu Phe Ly - #s Ala His Met Gly Gln      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Lys His Arg Ala Met Arg Val Ile Phe Ala Va - #l Val Leu Ile Phe        Leu                                                                                             245  - #               250  - #               255             - - Leu Cys Trp Leu Pro Tyr Asn Leu Val Leu Le - #u Ala Asp Thr Leu Met                  260      - #           265      - #           270                  - - Arg Thr Gln Val Ile Gln Glu Thr Cys Glu Ar - #g Arg Asn His Ile Asp              275          - #       280          - #       285                      - - Arg Ala Leu Asp Ala Thr Glu Ile Leu Gly Il - #e Leu His Ser Cys Leu          290              - #   295              - #   300                          - - Asn Pro Leu Ile Tyr Ala Phe Ile Gly Gln Ly - #s Phe Arg His Gly Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Leu Lys Ile Leu Ala Ile His Gly Leu Ile Se - #r Lys Asp Ser Leu        Pro                                                                                             325  - #               330  - #               335             - - Lys Asp Ser Arg Pro Ser Phe Val Gly Ser Se - #r Ser Gly His Thr Ser                  340      - #           345      - #           350                  - - Thr Thr Leu                                                                      355                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (synthetic)                                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CGCTCGAGAC CTRKCMDTKK CYGACCT          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (synthetic)                                   - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GCGAATTCTG GACRATGGCC AGGTAVCKGT C        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Asn Leu Ala Ile Ser Asp Leu                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Asp Arg Tyr Leu Ala Ile Val                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Ar - #g Tyr Leu Ala Ile Val      1               5   - #                10  - #                15               - - His Ala Val Phe Ala Leu Lys Ala Arg Thr Va - #l Thr Phe Gly Val                      20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Ar - #g Tyr Leu Ala Ile Val       1               5  - #                 10 - #                 15              - - His Ala Val Phe Ala Leu Arg Ala Arg Thr Va - #l Thr Phe Gly Val                      20      - #            25      - #            30                 __________________________________________________________________________

We claim:
 1. An isolated polypeptide comprising the amino acid sequenceof an MCP-1R polypeptide which is(a) a polypeptide having a sequence atleast 90% identical to the sequence of MCP-1RA as shown in SEQ ID NO: 2,(b) a polypeptide having a sequence at least 90% identical to thesequence of MCP-1RB as shown in SEQ ID NO: 4, or (c) a fragment of apolypeptide according to (a) or (b) which binds specifically to MCP-1,wherein the sequence of the fragment is at least 90% identical to thecorresponding portion of SEQ ID NO: 2 or SEQ ID NO: 4;wherein the MCP-1Rpolypeptide binds specifically to MCP-1 but not to MIP-1α, MIP-1β,RANTES, or IL-8 under physiological conditions.
 2. An isolatedpolypeptide according to claim 1, wherein the polypeptides (a) and (b)and the fragment (c) are at least 95% identical to SEQ ID NO: 2, SEQ IDNO: 4, and the corresponding portion of SEQ ID NO: 2 or SEQ ID NO: 4,respectively.
 3. An isolated polypeptide according to claim 1,comprising the amino acid sequence of an MCP-1R polypeptide having asequence at least 90% identical to the sequence shown in SEQ ID NO: 2 orSEQ ID NO:
 4. 4. An isolated polypeptide according to claim 3,comprising the amino acid sequence of an MCP-1R polypeptide having asequence at least 95% identical to the sequence shown in SEQ ID NO: 2 orSEQ ID NO:
 4. 5. An isolated polypeptide according to any one of claims1 to 4, free of association with other polypeptides.
 6. A polypeptidefree of association with other polypeptides, wherein the polypeptidecomprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.7. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding the amino acid sequence of an MCP-1R polypeptide according toclaim
 1. 8. An isolated nucleic acid molecule comprising a nucleotidesequence encoding the amino acid sequence of an MCP-1R polypeptideaccording to claim
 2. 9. A nucleic acid molecule according to claim 8,wherein the polypeptides (a) and (b) have the amino acid sequences shownin SEQ ID NOs: 2 and 4, respectively.
 10. An isolated nucleic acidmolecule comprising a nucleotide sequence encoding the amino acidsequence of an MCP-1R polypeptide according to claim
 3. 11. An isolatednucleic acid molecule comprising a nucleotide sequence encoding theamino acid sequence of an MCP-1R polypeptide according to claim
 4. 12.An isolated nucleic acid molecule comprising a nucleotide sequenceencoding the amino acid sequence shown in SEQ ID NO:
 2. 13. A nucleicacid molecule according to claim 12, comprising the nucleotide sequenceshown as residues 50 to 1161 of SEQ ID NO:
 1. 14. A nucleic acidmolecule according to claim 13, comprising the entire nucleotidesequence shown in SEQ ID NO:
 1. 15. An isolated nucleic acid moleculecomprising a nucleotide sequence encoding the amino acid sequence shownin SEQ ID NO:
 4. 16. A nucleic acid molecule according to claim 15,comprising the nucleotide sequence shown as residues 81 to 1160 of SEQID NO:
 3. 17. A nucleic acid molecule according to claim 16, comprisingthe entire nucleotide sequence shown in SEQ ID NO:
 3. 18. An isolatednucleic acid molecule encoding an MCP-1R polypeptide, wherein thenucleic acid molecule comprises(a) the nucleotide sequence of an MCP-1RcDNA that is present in a mammalian library and that hybridizes understringent conditions with a probe having the sequence of the complementof the coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, whereinthe cDNA encodes an MCP-1R polypeptide that binds specifically to MCP-1but not to MIP-1α, MIP-1β, RANTES, or IL-8 under physiologicalconditions; (b) the nucleotide sequence of a fragment of an MCP-1R cDNAaccording to (a), wherein a polypeptide having the MCP-1R amino acidsequence encoded by the fragment binds specifically to MCP-1; or (c) anucleotide sequence degenerate with (a) or (b).
 19. A nucleic acidmolecule according to claim 18, comprising the nucleotide sequence ofsaid MCP-1R cDNA.
 20. An expression vector comprising the nucleotidesequence of a nucleic acid molecule according to any one of claims 7 to19.
 21. A mammalian, insect, or yeast cell comprising heterologousnucleic acid having the sequence of a molecule according to any one ofclaims 7 to 19, wherein the cell is capable of effecting replication andexpression of the heterologous nucleic acid.
 22. A cell according toclaim 21, wherein the MCP-1R polypeptide encoded by the heterologousnucleic acid is stably expressed on the surface of the cell.
 23. Aprocess for producing an MCP-1R polypeptide, comprising culturing a cellaccording to claim 21 under conditions suitable to effect expression ofthe heterologous nucleic acid.
 24. A process according to claim 23,further comprising the step of isolating the expressed MCP-1Rpolypeptide from the cell.
 25. A method to identify an agonist orantagonist of MCP-1R, comprising:(a) contacting a cell according toclaim 22 with MCP-1 in the presence and absence of a candidate organiccompound, and (b) comparing the binding of the MCP-1 to the MCP-1Rpolypeptide in the presence of the compound to the binding of the MCP-1to the MCP-1R polypeptide in its absence;wherein a compound which causesan increase in the binding of MCP-1 to the MCP-1R polypeptide isidentified as an agonist of MCP-1R, and a compound which causes adecrease in the binding of MCP-1 to the MCP-1R polypeptide is identifiedas an antagonist of MCP-1R.
 26. A method according to claim 25, whereinthe compound causes a decrease in the binding of MCP-1 to the MCP-1Rpolypeptide.
 27. A method according to claim 25, wherein the MCP-1 isdetectably labeled.
 28. A method according to claim 25, wherein thebinding of MCP-1 to the MCP-1R polypeptide is determined in animmunoassay.